Drug screening and treatment methods

ABSTRACT

The invention relates to methods identify or characterize compounds that can be used to treat specified clinical disorders such as hyperglycemia and type 2 diabetes. Compounds that can be used in these methods include 4α-fluoro-17α-ethynylandrost-5-ene-3β,7β,17β-triol, 4α-fluoro-17α-ethynylandrost-5-ene-3β,7α,17β-triol, 4α-fluoro-17α-ethynylandrost-5-ene-3α,7β,17β-triol and 4α-fluoro-17α-ethynylandrost-5-ene-3β,17β-triol-7-one.

CROSS REFERENCE TO RELATED APPLICATIONS

This nonprovisional U.S. patent application is a continuation-in-part ofand claims priority from pending U.S. application Ser. No. 11/941,934,filed Nov. 17, 2007, and claims priority from U.S. provisionalapplication Ser. No. 60/866,395, filed Nov. 17, 2006, U.S. provisionalapplication Ser. No. 60/866,700, filed Nov. 21, 2006, U.S. provisionalapplication Ser. No. 60/868,042, filed Nov. 30, 2006, U.S. provisionalapplication Ser. No. 60/885,003, filed Jan. 15, 2007, and U.S.provisional application Ser. No. 60/888,058, filed Feb. 2, 2007, all ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods and compounds such as4α-fluoro-17α-ethynylandrost-5-ene-3β,7β,17β-triol to modulateinflammation, metabolic disorders and other conditions described herein.The compounds can be used to treat or slow the progression of conditionssuch as type 2 diabetes, hyperglycemia and insulin resistance.

BACKGROUND OF THE INVENTION

A number of factors contribute to the establishment and maintenance ofmany chronic autoimmune and inflammation disorders. Often, the etiologyof such disorders is not well understood. Tumor necrosis factor-α (TNFα)is a cytokine that is released primarily by mononuclear phagocytes inresponse to a number immunostimulators. When administered to animals orhumans, it causes inflammation, fever, cardiovascular effects,hemorrhage, coagulation, and acute phase responses similar to those seenduring acute infections and shock states. Excessive or unregulated TNFαproduction is thus implicated in a number of disease conditions. Theseinclude endotoxemia and/or toxic shock syndrome, e.g., Tracey et al.,Nature 330:662-664 (1987) and Hinshaw et al., Circ. Shock 30:279-292(1990), cachexia, e.g., Dezube et al., Lancet, 335(8690):662 (1990) andARDS where high TNFα concentrations have been detected in pulmonaryaspirates from ARDS patients, e.g., Millar et al., Lancet2(8665):712-714 (1989).

TNFα also may be involved in bone resorption diseases, includingarthritis. When activated, leukocytes can produce bone-resorption, anactivity to which TNFα may contribute, e.g., Bertolini et al., Nature319:516-518 (1986) and Johnson et al., Endocrinology 124(3):1424-1427(1989). TNFα also has been shown to stimulate bone resorption andinhibit bone formation in vitro and in vivo through stimulation ofosteoclast formation and activation combined with inhibition ofosteoblast function. Blocking TNFα with monoclonal anti-TNFα antibodieshas been shown to be beneficial in rheumatoid arthritis (Elliot et al.,Int. J. Pharmac. 17(2):141-145 1995) and Crohn's disease (von Dullemenet al., Gastroenterology, 109(1):129-135 2005).

The nuclear factor-kappaB (NF-κB) molecule is a mediator of inflammationin a number of clinical conditions. Some therapeutic agents that areused to treat inflammation such as dexamethasone, prednisone orhydrocortisol are glucocorticoid receptor (GR) agonists and theyindirectly inhibit NF-κB by increasing the activity of the GR, e.g., H.Harkonarson et al., Am. J. Respir. Cell Mol. Biol. 25:761-771, 2001.However, elevated levels of natural GR agonists and pharmacologicallevels of synthetic GR agonists usually exert unwanted toxicitiesincluding significant immune suppression and loss of bone mass orosteopenia, e.g., T. L. Popper et al., Anti-inflammatory agents:Anti-inflammatory steroids, R A. Scherer & M. W. Whitehouse, editors,Academic Press, New York, Chapter 9, volume 1, pages 245-294, 1974. Manyof the unwanted toxicities associated with glucocorticoids are caused byactivation of the GR. Thus, Identification of compounds that can inhibitNF-κB activity without causing these toxicities by activating the GRrepresents a class of agents that could be used to treat inflammationand associated symptoms such as pain, fever or fatigue.

Unwanted or damaging inflammation occurs in a number of chronic or acuteconditions, e.g., ARDS, COPD and sepsis. Activated monocytes andneutrophils may play a role in mediating inflammation associatedpathology in some of these conditions. Activated neutrophils can haveincreased NFκ-B in the nucleus and increased production ofproinflammatory cytokines. Neutrophils can be a source of toxic oxygenspecies whose generation mediates, at least in part, tumor necrosisfactor-alpha (TNF-α) secretion by activated macrophages. TNFα may benecessary for some of the organ injury and failure that can be seen insepsis.

Signaling associated with inflammation can occur through differentpathways and this can increase the activity of NF-κB in affected cells.NF-κB activation by tumor necrosis factor-α (TNF-α) starts with bindingof TNF-α to the TNF-α receptor at the cell membrane, followed byactivation of a series on signal transducers including MAP kinases.Activation of NF-κB in the cytoplasm leads to its translocation into thenucleus and activation of genes that contain the NF-κB response elementin their promoters. Activation of cytoplasmic NF-κB by bacteriallipopolysaccharide (LPS) begins with binding of LPS to Toll-likereceptor 4 at the cell surface and subsequent activation ofintracellular signal transducers, includingphosphatidylinositol-3-kinase. TNF-α and LPS are both known to induceintense inflammatory responses in vivo and in cells in vitro. Cells thatrespond to such proinflammatory signals include macrophages, monocytesand other types of immune cells.

Various T cell subsets appear to have a role in the development ofcertain disease conditions. An important role for a distinct T cellpopulations including regulatory and/or suppressor T cells in mediatingvarious aspects of immunity has been suggested, e.g., E. Suri-Payer etal., J. Immunol., 160(3): 1212-1218, 1998; J. Shimizu et al., J.Immunol., 163(10):5211-5218, 1999; M. Itoh et al., J. Immunol.,162(9):5317-5326, 1999; A. M. Miller et al., J. Immunol., 177:7398-7405,2006. CD4⁺ CD25⁺ T cells may play a role in suppressing some immuneresponses.

Study of some of these T cell subsets in animal models have beendescribed, e.g., U.S. Pat. No. 6,593,511. For example, a role for thestudy of human autoimmune conditions was examined in the scid/scid CD4⁺CD45Rb^(hi) model. This animal model has been used to study dysregulatedimmune responses such as inflammation conditions and to evaluateexperimental drugs and treatment protocols, e.g., K. Hong et al., J.Immunol., 162:7480-7491, 1999; Powrie et al., J. Exp. Med.,183(6):2669-2674, 1996.

The Foxpro3 gene, which is induced by thymus epithelium may play a rolein inducing T cells to develop the CD4⁺CD25⁺ or CD4⁺CD25^(high) (Treg orregulator T cell) phenotype. The CD25 surface antigen is the IL-2receptor α-chain. In some animal models of autoimmune diseases,deficiency of the Foxpro3 gene is associated with the occurrence ofautoimmune diseases, e.g., U.S. patent application No. 2006/0111316.Restoration of this gene appears to reduce autoimmune anomalies. Variousreagents or assay protocols for CD4⁺CD25⁺ cells have been described,e.g., H. Yagi et al., International Immunol., 16(11):1643-1656, 2004; W.R. Godfrey et al., Blood, 105(2)750-758, 2005.

Insulin resistance in glucose intolerant subjects has long beenrecognized. Reaven et al (American Journal of Medicine, 60(1):80-88,1976) used a continuous infusion of glucose and insulin (insulin/glucoseclamp technique) and oral glucose tolerance tests to demonstrate thatinsulin resistance existed in a diverse group of nonobese, nonketoticsubjects. These subjects ranged from borderline glucose tolerant toovert, fasting hyperglycemia. The diabetic groups in these studiesincluded both insulin dependent (IDDM) and noninsulin dependent (NIDDM)subjects.

Coincident with sustained insulin resistance is the more easilydetermined hyperinsulinemia, which can be measured by accuratedetermination of circulating plasma insulin concentration in the plasmaof subjects. Hyperinsulinemia can be present as a result of insulinresistance, such as is in obese and/or diabetic (NIDDM) subjects and/orglucose intolerant subjects, or in IDDM subjects, as a consequence ofover injection of insulin compared with normal physiological release ofthe hormone by the endocrine pancreas.

The association of hyperinsulinemia with obesity and with ischemicdiseases of the large blood vessels (e.g. atherosclerosis) has beendescribed by experimental, clinical and epidemiological studies (Stout,Metabolism, 34:7, 1985; Pyorala et al, Diabetes/Metabolism Reviews,3:463, 1987). Statistically significant plasma insulin elevations at 1and 2 hours after oral glucose load correlate with an increased risk ofcoronary heart disease.

One model of human diabetes is the db/db mouse. The db/db mouse modelhas been described, e.g., D. Koya et al., The FASEB Journal, 14:439-447,2000; K. Kobayashi et al., Metabolism, 49(1): 22-31, 2000; J. Berger etal., J. Biol. Chem., 274(10):6718-6725, 1999. The db/db mice carry amutation in the gene encoding the leptin receptor, which confers aphenotype characterized by hyperphagia, obesity, insulin resistance anddiabetes as their functional pancreatic β-cell mass deteriorates overtime, particularly for animals in the C57BL/Ks genetic background. Thedb/db mice typically become identifiably obese at around 3 to 4 weeks ofage and elevations of plasma insulin begin at 10 to 14 days. Elevationsof blood sugar are seen at 4 to 8 weeks of age with an uncontrolled risein blood sugar, severe depletion of the insulin producing β-cells of thepancreatic islets, and death by about 10 months of age. This model hasbeen used to characterize the capacity of drug candidates to affect theonset or rate of progression of parameters, e.g., hyperglycemia andweight gain, related to the development and maintenance of diabetes.

Treatment of diabetes with PPAR-γ agonists has been associated withcardiac hypertrophy, or an increase in heart weight. Treatment withrosiglitazone maleate, a PPAR-γ agonist, indicate that patients mayexperience fluid accumulation and volume-related events such as edemaand congestive heart failure. Cardiac hypertrophy related to PPAR-γagonist treatment is typically treated by discontinuing the treatment.

A physiological effect of cortisol is its antagonism to insulin. Highcortisol concentrations in the liver can reduce insulin sensitivity inthat organ, which tends to increase gluconeogenesis and increase bloodsugar levels (M. F. Dallman et al. Front Neuroendocrinol., 14:303-347,1993). This effect aggravates impaired glucose tolerance or diabetesmellitus. In Cushing's syndrome, which is caused by excessivecirculating concentrations of cortisol, the antagonism of insulin canprovoke diabetes mellitus in susceptible individuals (E. J. Ross et al.,Lancet, 2:646-649, 1982).

Cortisol can be converted in the body to cortisone by the11b-dehydrogenase activity of 11b-hydroxysteroid dehydrogenase enzymes.The reverse reaction, converting inactive cortisone to active cortisol,is accomplished in certain organs by the 11b-reductase activity of theseenzymes. This activity is also known as corticosteroid 11b-reductaseactivity. There are at least two distinct isozymes of 11β-hydroxysteroiddehydrogenase. Expression of 11β-HSD type 1 in a range of cell linesgenerates either a bi-directional enzyme or a predominant 11β-reductase,which can regenerate 11β-hydroxysteroid from the otherwise inert 11-ketosteroid parent.

Mitochondrial phosphoenolpyruvate carboxykinase (also known asPEPCK-mitochondrial, PEPCK-M, PCK2 and mtPEPCK) is expressed in avariety of human tissues, mainly the liver, kidney, pancreas, intestineand fibroblasts (Modaressi et al., Biochem. J., 333:359-366, 1998).PEPCK-mitochondrial deficiency, while not well documented, has beenassociated with failure to thrive, hypoglycemia and liver abnormalities.Unlike the cytosolic form (PEPCK-C), the mitochondrial form(PEPCK-mitochondrial) is expressed constitutively and is not regulatedby hormonal stimuli (Hanson and Patel, Adv. Enzymol. Relat. Areas Mol.Biol., 69:203-281, 1994). The two forms are located on separatechromosomes with localized to chromosome 14q11 and PEPCK-C resides onchromosome 20q11 (Stoffel et al., Hum. Mol. Genet. 2:1-4, 1993).

Multiple sclerosis (MS) is an autoimmune disease that is an inflammatorydisease of the central nervous system (Bar-Or, A., J. Neuroimmunol.100:252-259, 1999). Although the natural course of the disease hasrecently been improved by treatment withimmunomodulatory-immunosuppressive compounds such as Interferon(IFN)-beta, copolymer, cyclophosphamide and mitoxantrone (Hafler, D. A.and Weiner, H. L., Immunological Reviews 144:75, 1995; Goodkin, D. E.,Lancet 352: 1486, 1998), none of these drugs can block progression ofdisease and some of them have serious side-effects that limit theirprolonged use. In addition, a substantial number of patients with bothrelapsing-remitting and secondary progressive MS exhibit poor responseto IFN-β. Therefore, there is a need for novel compounds that alone orin combination therapy improve the course of MS by e.g., slowing itsprogression.

There is a current need for cost-effective pharmaceutical agents ortreatment methods that are more effective in treating conditionsdescribed herein. The present invention provides therapeutic agents andtreatment methods to treat one or more of the conditions describedherein. The claimed agents and methods are useful to reduce one or moresymptoms associated with the conditions described herein. Also, the useof the invention agents and methods can be combined with one or moreconventional treatments for these disorders.

DESCRIPTION OF THE INVENTION Summary of Invention Embodiments

Invention embodiments include a method to identify a compound with apotential to treat or ameliorate a metabolic disorder in a mammal,comprising selecting a compound that; (i) does not activate one, two orthree of PPAR-α, PPAR-γ and PPAR-δ in human or mammalian cells in vitroby more than about 30% when compared to suitable negative control humanor mammalian cells in vitro; (ii) inhibits or decreases thetranscriptional activity or level of NF-κB by about 20-80% in human ormammalian cells in vitro when compared to suitable negative controlhuman or mammalian cells in vitro; (iii) when compared to a suitablenegative control or normal control, (a) decreases the degree ofhyperglycemia, (b) slows the progression of hyperglycemia, (c) delaysthe onset of hyperglycemia, (d) decreases the rate of maculardegeneration, (e) delays the onset of macular degeneration, (f)decreases the occurrence or incidence of vascular ulcers, (g) decreasesthe severity of vascular ulcers, (h) increases insulin sensitivity, (i)decreases glucose intolerance, (j) slows the progression or rate of lossof pancreatic β-islet cell numbers or their capacity to secrete insulin,(k) increases pancreatic β-islet cell numbers or their capacity tosecrete insulin, (l) slows the rate of weight increase in db/db mice ormice with diet induced obesity, (m) decreases elevated levels oftriglycerides, (n) decreases elevated levels total blood or serumcholesterol, (o) decreases normal or elevated levels of LDL, VLDL,apoB-100 or apoB-48 in blood or serum, (p) increases normal or lowlevels of HDL or apoA1 in blood or serum, (q) decreases or normalizes anelevated level of a phase reactive protein, optionally C reactiveprotein or fibrinogen in blood or serum, (r) decreases or normalizeshemoglobin A_(1C), (s) decreases or normalizes fasting blood glucoselevels, (t) normalizes serum or blood glucose in an oral glucosetolerance test, (u) normalizes serum or blood glucose in a 2 hour postprandial glucose test or (v) increases whole body or tissue glucosedisposal or uptake in a human or another mammal in vivo, for 1, 2 or 3of the foregoing; (iv) optionally, does not activate one or more of aglucocorticoid receptor, an androgen receptor an estrogen receptor-α,estrogen receptor-β or a biologically active variant of any of thesebiomolecules in human or mammalian cells in vitro by more than about 30%when compared to suitable negative control human or mammalian cells invitro; and (v) optionally inhibits the level or activity ofphosphoenolpyruvate carboxykinase (PEPCK) or a 11β-hydroxysteroiddehydrogenase (11β-HSD), optionally 11β-HSD type 1 or 11β-HSD type 2 orthe level of a mRNA that encodes PEPCK or a 11β-HSD, in hepatocytes orliver-derived cells in vitro or in liver cells or tissue obtained fromliver cells or tissue in vivo; optionally whereby the compound with apotential to treat or ameliorate the metabolic disorder in a mammal isidentified and optionally recorded as such in a written or a readableelectronic medium. In some embodiments, the compound accomplishes one ortwo of, e.g., a decrease in the degree of hyperglycemia, improvesinsulin resistance, a decrease or normalization of fasting blood glucoselevels, e.g., in obese, diabetic or pre-diabetic patients or subjects,an increase in whole body or tissue glucose disposal or uptake, e.g., ina rodent or human or amelioration of diabetes as observed by improvementin one or more symptoms associated with type 2 diabetes or ahyperlipidemia condition described herein.

In other embodiments, the invention provides a method to treat apulmonary condition, e.g., such as asthma, cystic fibrosis, lungfibrosis, acute respiratory distress syndrome (ARDS) or chronicobstructive pulmonary disease (COPD), in a mammal having the pulmonarycondition comprising administering an effective amount of a compoundhaving the structure

wherein the dotted lines are an optional double bond and if one ispresent, the hydrogen atom at the 5-position is absent, or hydrogen ispresent in the α- or β-configuration; wherein one R¹ is —H or C₁₋₈optionally substituted alkyl and the other R¹ is —OH, an ester or anether; one R² is —H or C₁₋₈ optionally substituted alkyl and the otherR² is —OH or an ester, or both R² together are ═O; one R³ is —H and theother R³ is —H, —OH, an ester or an ether; R⁴ in the α-configuration isoptionally substituted alkynyl such as optionally substituted C₂₋₄alkynyl; R⁴ in the β-configuration is —OH or an ester; R⁷ is —CH₃, —C₂H₅or —CH₂OH; one R¹¹ is —H or C₁₋₈ optionally substituted alkyl and theother R¹¹ is —H, —OH or an ester, or both R¹¹ together are ═O or ═NOH;R¹⁵ is —H, —OH, halogen, an ester or an ether in the α-configuration orthe β-configuration or ═O if no double bond is present at the 4-5position or R¹⁵ is —H, an ester or an ether if a double bond is presentat the 4-5 position; and R¹⁶ is —H, —OH, an ester or an ether in theα-configuration or the β-configuration or ═O or ═NOH.

The invention provides compounds or compositions to treat, prevent, slowthe progression of or ameliorate an unwanted inflammation condition anautoimmune disease or a metabolic disorder or a symptom thereof, in amammal in need thereof, optionally wherein the mammal is a human or anon-human primate, optionally wherein the compositions comprise aformula 1 compound (“F1C”) having the structure

wherein, the dotted lines are optional double bonds, one R¹ is —H or acarbon-linked moiety such as optionally substituted alkyl and the otherR¹ is an oxygen-linked moiety, a sulfur-linked moiety or anitrogen-linked moiety, or both R¹ together are ═O, ═NOH or ═NO—C₁₋₆alkyl; one R² is —H or a carbon-linked moiety such as optionallysubstituted alkyl and the other R² is —H, an oxygen-linked moiety, asulfur-linked moiety or a nitrogen-linked moiety, or both R² togetherare ═O; one R³ is —H or a carbon-linked moiety such as optionallysubstituted alkyl and the other R³ is —H, an oxygen-linked moiety, asulfur-linked moiety or a nitrogen-linked moiety; one R⁴ is —H or acarbon-linked moiety such as optionally substituted alkyl and the otherR⁴ is an oxygen-linked moiety, a sulfur-linked moiety or anitrogen-linked moiety, or both R⁴ together are ═O, ═NOH or ═NO—C₁₋₆alkyl; one R⁵ is —H or a carbon-linked moiety such as optionallysubstituted alkyl and the other R⁵ is —H, an oxygen-linked moiety, asulfur-linked moiety or a nitrogen-linked moiety; R⁶ is —H or C₁₋₆optionally substituted alkyl, optionally —CH₃; and R⁷ is —H or C₁₋₆optionally substituted alkyl, optionally —CH₃, —CH₂OH or —C₂H₅; R⁸ is—CH₂—, or —C(R¹⁰)₂— where R¹⁰ independently or together are —H, ═O, acarbon-linked moiety such as optionally substituted alkyl, anoxygen-linked moiety, optionally —OH or an ester or ether optionallyselected from —OC(O)—CH₃, —OC(O)—C₂H₅, —OCH₃ and —OC₂H₅, a sulfur-linkedmoiety or a nitrogen-linked moiety; and R⁹ is —CH₂—, or —C(R¹⁰)₂— whereR¹⁰ independently or together are —H, halogen, ═O, a carbon-linkedmoiety such as optionally substituted alkyl, an oxygen-linked moiety,optionally —OH or an ester or ether optionally selected from —OC(O)—CH₃,—OC(O)—C₂H₅, —OCH₃ and —OC₂H₅, a sulfur-linked moiety or anitrogen-linked moiety.

The invention also provides a method to identify or characterize abiological activity of a compound with a potential to treat orameliorate a metabolic disorder in a mammal, comprising selecting acompound that (i) does not activate one, two or three of PPAR-α, PPAR-γand PPAR-δ in human or mammalian cells in vitro by more than about 30%when compared to suitable negative control human or mammalian cells invitro; (ii) inhibits or decreases the transcriptional activity or levelof NF-κB by about 20-80% in human or mammalian cells in vitro whencompared to suitable negative control human or mammalian cells in vitro;(iii) when compared to a suitable negative control or normal control,decreases hyperglycemia, slows the progression or delays the onset ofhyperglycemia, increases insulin sensitivity, decreases glucoseintolerance, slows the progression or rate of loss of pancreatic β-isletcell numbers or their capacity to secrete insulin, increases pancreaticβ-islet cell numbers or their capacity to secrete insulin, slows therate of weight increase in db/db mice or in subjects with diet inducedor diet related obesity, decreases elevated levels of triglycerides,decreases elevated levels total blood or serum cholesterol, decreasesnormal or elevated levels of LDL, VLDL, apoB-100 or apoB-48 in blood orserum or increases normal or low levels of HDL or apoA1 in blood orserum or decreases an elevated level of fibrinogen in blood or serum;and (iv) optionally, does not activate one or more of a glucocorticoidreceptor, a mineralcorticoid receptor, a progesterone receptor, anandrogen receptor an estrogen receptor-α, estrogen receptor-β or abiologically active variant of any of these biomolecules in human ormammalian cells in vitro by more than about 30% when compared tosuitable negative control human or mammalian cells in vitro. The methodallows identification or characterization of the compound as having apotential to treat or ameliorate the metabolic disorder in human oranother mammal.

Embodiments also include compositions comprising a formula 1 compoundfor the prophylaxis or treatment of an autoimmune condition, an unwantedinflammation condition or a metabolic disorder or a symptom of any ofthese conditions.

Other embodiments are as described elsewhere in the specificationincluding the embodiments described herein.

DEFINITIONS

As used herein and unless otherwise stated or implied by context, termsthat are used herein have the meanings that are defined here. Thedescriptions of embodiments and examples that are described illustratethe invention and they are not intended to limit it in any way. Unlessotherwise contraindicated or implied, e.g., by including mutuallyexclusive elements or options, in these definitions and throughout thisspecification, the terms “a” and “an” mean one or more and the term “or”means and/or.

The phrase “metabolic disorder” or “metabolic disease” means one or moreconditions such as type 1 diabetes, type 2 diabetes, obesity, insulinresistance, hyperglycemia, impaired glucose utilization or tolerance,impaired or reduced insulin synthesis, a hyperlipidemia condition suchas hypercholesterolemia, hypertriglyceridemia or elevated free fattyacids and hypolipidemia conditions. Hypercholesterolemias includehyper-LDL cholesterolemia or elevated LDL cholesterol. Hypolipidemiasinclude hypo-HDL cholesterolemia or low HDL cholesterol levels. Type 1diabetes includes Immune-Mediated Diabetes Mellitus and IdiopathicDiabetes Mellitus. Type 2 diabetes includes forms with predominant orprofound insulin resistance, predominant insulin deficiency and someinsulin resistance and forms intermediate between these. Otherdescriptions are elsewhere herein.

A “formulation” or the like means a composition that one can administerto a subject, e.g., human or animal. Formulations are suitable for humanor veterinary applications and would typically have expectedcharacteristics for the formulation, e.g., parenteral formulations forhuman use would usually be sterile solutions or suspensions.

An “excipient”, “carrier”, “pharmaceutically acceptable carrier” orsimilar terms mean one or more component(s) or ingredient(s) that isacceptable in the sense of being compatible with the other ingredientsof invention compositions or formulations and not overly deleterious tothe patient, animal, tissues or cells to which the formulation is to beadministered.

A “subject” means a human or animal. Usually the animal is a mammal orsuch as a non-human primate, rodent, lagomorph, domestic animal or gameanimal. Primates include chimpanzees, cynomologus monkeys, spidermonkeys, and macaques, e.g., Rhesus or Pan. Rodents and lagomorphsinclude mice, rats, woodchucks, ferrets, rabbits and hamsters.

“Alkyl” as used here means linked normal, secondary, tertiary or cycliccarbon atoms, i.e., linear, branched, cyclic or any combination thereof.Alkyl moieties, as used herein, may be saturated, or unsaturated, i.e.,the moiety may comprise one, two or more independently selected doublebonds or triple bonds. Unsaturated alkyl moieties include moieties asdescribed for alkenyl and alkynyl moieties described below. The numberof carbon atoms in an alkyl group or moiety is 1 to about 30, e.g.,about 1-20 or about 1-8, unless otherwise specified. Thus C₁₋₈ alkylmeans an alkyl moiety containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms.When an alkyl group is specified, species may include methyl, ethyl,1-propyl (n-propyl), 2-propyl (i-propyl, —CH(CH₃)₂), 1-butyl (n-butyl),2-methyl-1-propyl (i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-butyl, —C(CH₃)₃),—(CH₂)_(n)—(CHCH₃)_(m)—(CH₂)_(o)—CH₃ and—(CH₂)_(n)—(CHC₂H₅)_(m)—(CH₂)_(o)—CH₃ where n, m and o independently are0, 1, 2, 3, 4, 5, 6, 7 or 8.

“Alkenyl” as used here means a moiety that comprises linked normal,secondary, tertiary or cyclic carbon atoms, i.e., linear, branched,cyclic or any combination thereof, that comprises one or more doublebonds (e.g., —CH═CH—), e.g., 1, 2, 3, 4, 5, 6 or more, typically 1 or 2.The number of carbon atoms in an alkenyl group or moiety is 2 to about30, e.g., about 2-20 or about 2-8, unless otherwise specified, e.g.,C₂₋₈ alkenyl or C2-8 alkenyl means an alkenyl moiety containing 2, 3, 4,5, 6, 7 or 8 carbon atoms. When an alkenyl group is specified, speciesmay include vinyl, allyl, —(CH₂)_(n)—(CH═CH)—(CH₂)_(m)—CH₃,—(CH₂)_(n)—(CCH₃═CH)—(CH₂)_(m)—CH₃, —(CH₂)_(n)—(CH═CCH₃)—(CH₂)_(m)—CH₃and —(CH₂)_(n)—(CH═CH)₀₋₁—(CH₂)_(m)—CH₂CH═CH₂, where n and mindependently are 0, 1, 2, 3, 4, 5, 6, 7 or 8.

“Alkynyl” as used here means a moiety that comprises linked normal,secondary, tertiary or cyclic carbon atoms, i.e., linear, branched,cyclic or any combination thereof, that comprises one or more triplebonds (—C≡C—), e.g., 1, 2, 3, 4, 5, 6 or more, typically 1 or 2 triplebonds, optionally comprising 1, 2, 3, 4, 5, 6 or more double bonds, withthe remaining bonds being single bonds. The number of carbon atoms in analkenyl group or moiety is 2 to about 30, e.g., about 2-20 or about 2-8,unless otherwise specified, e.g., C₂₋₈ alkynyl or C₂₋₈ alkynyl means analkynyl moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms. When analkynyl group is specified, groups and species may include —CCH, —CCCH₃,—CCCH₂CH₃, —CCC₃H₇, —CCCH₂C₃H₇, —(CH₂)_(n)—(C≡C)—(CH₂)_(m)—CH₃, and—(CH₂)_(n)—(C≡C)₀₋₁—(CH₂)_(m)—CH₂C≡CH, where n and m independently are0, 1, 2, 3, 4, 5, 6, 7 or 8.

“Substituted alkyl”, “substituted alkenyl” “substituted alkynyl” and thelike mean an alkyl, alkenyl, alkynyl or another group or moiety asdefined herein that has a substituent(s) or that comprises asubstituent(s) that replaces a hydrogen atom(s) and is bonded to acarbon atom(s) or a substituent(s) that interrupts a carbon atom chain.Substituents include 1, 2, 3, 4, 5, 6 or more independently selected —F,—Cl, —Br, —I, —OH, —OR^(PR), —SH, —SCH₃, —O—, —S—, —NH—, —C(O)—,—C(O)OR^(PR), —CHO, —CH₂SH, —C═N—, —C(O)OR^(PR), —C(O)CH₃, where R^(PR)independently is hydrogen or a protecting group. Exemplary substitutedalkyl or substituted alkenyl moieties are —CCCl, —CH₂OH, —CF₃ and—CH₂(OH)CH₃.

“Halogen” means fluorine, chlorine, bromine or iodine.

“Ester” means a moiety that comprises a —C(O)—O— structure. Typically,esters as used here comprise an organic moiety containing about 1-50carbon atoms, usually about 2-20 or 2-8 carbon atoms and 0 to about 10independently selected heteroatoms (e.g., O, S, N, P, Si), where theorganic moiety is bonded to a formula 1 steroid nucleus at, e.g., R¹ orR² through the —C(O)—O— structure, e.g., organic moiety-C(O)—O-steroidor organic moiety-O—C(O)-steroid. The organic moiety usually comprisesone or more of any of the organic groups described above, e.g., C₁₋₈alkyl moieties, C₂₋₈ alkenyl moieties, C₂₋₈ alkynyl moieties, arylmoieties, C₂₋₉ heterocycles or substituted derivatives of any of these,e.g., comprising 1, 2, 3, 4 or more substituents, where each substituentis independently chosen. Esters include esters of succinic acid,dicarboxylic acids and amino acids such as —C(O)—(CH₂)_(n)—C(O)—OR^(PR),—O—C(O)—(CH₂)_(n)—NHR^(PR), —NH—(CH₂)_(n)—C(O)—OR^(PR), where n is 1, 2,3, 4, 5, 6, 7 or 8 and R^(PR) is —H or a protecting group such as C1-4alkyl. Esters also include structures such as —O—C(O)—O—(CH₂)_(n)—H and—O—C(O)—NH—(CH₂)_(n)—H.

Exemplary esters include one or more independently selected acetate,enanthate, propionate, isopropionate, cyclopropionate, isobutyrate,n-butyrate, valerate, caproate, isocaproate, hexanoate, heptanoate,octanoate, nonanoate, decanoate, undecanoate, phenylacetate or benzoate,which are typically hydroxyl esters. Esters also include amino acids,carbonates and carbamates including —O—C(O)—CH₂—NHR^(PR),—O—C(O)—CH₂CH₂—NHR^(PR), —O—C(O)—CH(CH₃)—NHR^(PR),—O—C(O)—CH₂CH(CH₃)—NHR^(PR), —O—C(O)—CH(NHR^(PR))—CH(OR^(PR))—CH₃,—O—C(O)—O—(CH₂)_(m)—H and —O—C(O)—NH—(CH₂)_(m)—H where R^(PR) is —H or aprotecting group such as C₁₋₄ alkyl (—CH₃, —C₂H₅, —C₃H₇, etc.) or—C(O)—CH₃ or —CH₂CH₂—O—CH₃ and m is 0, 1, 2, 3, 4, 5 or 6. Esters alsoinclude —O—C(O)—(CF₂)_(n)—CF₃, —O—C(O)—(CH₂)_(n)—CH₃,—O—C(O)—CH(CH₃)—(CH₂)_(n)—CH₃ and —O—C(O)—C(CH₃)₂—(CH₂)_(n)—CH₃ where nis 0, 1, 2, 3, 4, 5 or 6.

Preferred esters are —OC(O)CH₃, —OC(O)C₂H₅, —OC(O)—(CH₂)₂—CH₃,—OC(O)—(CH₂)₄—CH₃, —OC(O)—(CH₂)₁₀—CH₃, —OC(O)—(CH₂)₁₄—CH₃,—OC(O)—(CH₂)₁₆—CH₃, —OC(O)(CH₂)₇CH═CH—(CH₂)₇CH₃, with —OC(O)CH₃ and—OC(O)C₂H₅ usually being most preferred.

“Ether” means an organic moiety as described for ester that comprises 1,2, 3, 4 or more —O— moieties, usually 1 or 2. In some embodiments, the—O— group is linked to the steroid nucleus at a variable group such asR¹, R², R³, R⁴ or R¹¹, e.g., organic moiety-O-steroid. The organicmoiety is as described above for esters. Ethers include—O—(CH₂)_(n)—CH₃, —O—CH₂(CH₂)_(n)—O—CH₃, —O—CH₂(CH₂)_(n)—S—CH₃ and—O—CH(CH₃)—(CH₂)_(n)—CH₃ where n is 0, 1, 2, 3, 4, 5 or 6.

Formula 1 compounds. In some embodiments, the formula 1 compounds have3, 4 or 5 hydroxy groups, optionally wherein one, two or more areesterified with ester groups that are the same or different. Inpreferred embodiments, the 17-position is disubstituted with an oxygenlinked moiety such as —OH, ester or ether and either —H or a carbonlinked moiety, preferably optionally substituted C₂₋₄ alkynyl,preferably —CCH or —CC—Cl. In some of these embodiments the hydroxylgroups or esters are at the 3-, 4-, 16-, and 17-positions wherein thehydroxyl groups or esters at the 3-, 4- and 16-positions respectivelyare in the β,β,α, β,β,β, α,β,α, α,β,β, β,α,α, β,α,β, α,α,α or α,α,β,configurations. The hydroxyl or ester at the 17-position is typically inthe β-configuration, but can be in the α-configuration. R¹⁰ in thesecompounds is typically —H or a halogen such as —F and R⁵ optionally is—CH₃ or —C₂H₅ and R⁶ optionally is —H or —CH₃. For some of thesecompounds an additional hydroxyl or ester can be present at the7-position or the 11-position in the β-configuration or theα-configuration.

F1C structures include

wherein one R¹ is —H or optionally substituted alkyl and the other R¹ is—H, —OH, an ester or an ether, or both R¹ together are ═O or an oximesuch as ═NOH or ═NOCH₃; an ester or an ether; one R² is —H or optionallysubstituted alkyl and the other R² is —H, —OH, an ester or an ether orboth R² together are ═O or an oxime such as ═NOH or ═NOCH₃; one R³ is —Hor optionally substituted alkyl and the other R³ is —H, —OH, an ester oran ether or optionally substituted alkyl or both R³ together are ═O oran oxime such as ═NOH or ═NOCH₃; one R⁴ is —H or optionally substitutedalkyl and the other R⁴ is —OH, an ester or an ether or both R⁴ togetherare or both R⁴ together are ═O or an oxime such as ═NOH or ═NOCH₃; R⁵ isoptionally substituted alkyl, optionally selected from —CH₃, —C₂H₅ and—CH₂OH; R⁶ is —H or optionally substituted alkyl, optionally selectedfrom —CH₃, —C₂H₅ and —CH₂OH; one R⁷ is —H or optionally substitutedalkyl and the other R⁷ is —H, —OH, an ester or an ether or, when nodouble bond is present at the 4-position both R⁷ together are ═O or anoxime such as ═NOH or ═NOCH₃; R⁹ is —O— or —C(R¹²)(R¹²)— where one R¹²is —H, —F, —Br or optionally substituted alkyl and the other R¹² is —H,—OH, an ester or an ether or optionally substituted alkyl or both R¹²together are ═O or an oxime such as ═NOH or ═NOCH₃; R¹⁰ is —H or ahalogen such as —F or —Cl; and one R¹¹ is —H or optionally substitutedalkyl and the other R¹¹ is —H, —OH, an ester or an ether or optionallysubstituted alkyl or both R¹¹ together are ═O or an oxime such as ═NOHor ═NOCH₃. Embodiments of these compounds include compounds wherein (i)one, two or three of R², R⁷, R¹¹ and R¹² independently are —OH, a C₂₋₈ester, a C₁₋₈ ether or ═O, (ii) one or two of R¹, R⁷, R¹¹ and R¹² are—OH, a C₂₋₈ ester or a C₁₋₈ ether and (iii) one or two of R¹, R², R⁷,R¹¹ and R¹² are ═O or ═NOH and one, two or three of the othersindependently are —OH, a C₂₋₈ ester or a C₁₋₈ ether. The hydrogen atomat the 5-position, when present, can be in the α- or β-configuration.When a double bond is present at the 4-position, one R⁷ moiety isabsent.

For formula 1 compounds C₁₋₈ substituted alkyl moieties usually include—CH₂F, —CF₃, —CH₂OH and —C₂F₅. C₂₋₄ optionally substituted alkynylmoieties usually include —CCH, —CCCl, —CCCH₃, —CCCH₂OH, —CCCH₂Cl and—CCCH₂Br.

Exemplary formula 1 compounds include17α-ethynylandrost-5-ene-3β,4β,7β,16α,17β-pentol and epimers of thiscompound where one or two hydroxyl groups are epimerized, e.g.,17α-ethynylandrost-5-ene-3α,4β,7β,16α,17β-pentol,17α-ethynylandrost-5-ene-3β,4α,7α,16α,17β-pentol and17α-ethynylandrost-5-ene-3β,4β,7α,16β,17β-pentol. Other formula 1compounds include ones where 5 independently selected —OH, ester orether moieties are present, e.g.,17α-ethynylandrost-5-ene-3β,4β,11β,16α,17β-pentol and epimers of thiscompound where one or two hydroxyl groups are epimerized, e.g.,17α-ethynylandrost-5-ene-3α,4β,11β,16α,17β-pentol,17α-ethynylandrost-5-ene-3β,4β,11β,16α,17α-pentol,17α-ethynylandrost-5-ene-3β,4β,11α,16α,17β-pentol and17α-ethynylandrost-5-ene-3β,4α,11β,16β,17β-pentol. For such compounds,one, two or more of the 5 hydroxyl groups can be derivatized, e.g., toesters or ethers such as methoxy, ethoxy, acetoxy, propionoxy,—O—C(O)—(CH₂)₃—H, —O—C(O)—(CH₂)₄—H or —O—C(O)—(CH₂)₅—H derivatives.Other esters include —O—C(O)—CH₂—C(O)OH, —O—C(O)—(CH₂)₂—C(O)OH,—O—C(O)—(CH₂)₃—C(O)OH, —O—C(O)—(CH₂)₄—C(O)OH, —O—C(O)—(CH₂)₅—C(O)OH and—O—C(O)—(CH₂)₆—C(O)OH. Exemplary compounds include3β-acetoxy-17α-ethynylandrost-5-ene-4β,7β,16α,17β-tetrol,3β-acetoxy-17α-ethynylandrost-5-ene-4β,7α,16α,17β-tetrol,3α-acetoxy-17α-ethynylandrost-5-ene-4β,7β,16α,17β-tetrol,7β-acetoxy-17α-ethynylandrost-5-ene-3α,4β,16α,17β-tetrol,3β,7β-diacetoxy-17α-ethynylandrost-5-ene-4β,16α,17β-triol,3α,16α-diacetoxy-17α-ethynylandrost-5-ene-4β,7α,17β-triol,3β,16α-diacetoxy-17α-ethynylandrost-5-ene-4α,7α,17β-triol,3α,17β-diacetoxy-17α-ethynylandrost-5-ene-4β,7β,16α-triol,3β-acetoxy-17α-ethynylandrost-4-ene-4β,7β,16α,17β-tetrol,3β-acetoxy-17α-ethynylandrost-4-ene-4β,7α,16α,17β-tetrol,3α-acetoxy-17α-ethynylandrost-4-ene-4β,7β,16α,17β-tetrol,7β-acetoxy-17α-ethynylandrost-4-ene-3α,4β,16α,17β-tetrol,3β-acetoxy-17α-ethynylandrostane-4β,7β,16α,17β-tetrol,3β-acetoxy-17α-ethynylandrostane-4β,7α,16α,17β-tetrol,3α-acetoxy-17α-ethynylandrostane-4β,7β,16α,17β-tetrol and7β-acetoxy-17α-ethynylandrostane-3α,4β,16α,17β-tetrol.

In some of these embodiments four independently selected hydroxyl,esters or ethers are present in a formula 1 compound at four of the 2-,3-, 4-, 7-, 11-, 16-, and 17-positions. Such substituents can be bondedto, e.g., the 2-, 3-, 16-, and 17-positions, 2-, 3-, 7-, and17-positions, 2-, 3-, 11-, and 17-positions, 3-, 4-, 7-, and17-positions, 3-, 4-, 11-, and 17-positions, 3-, 7-, 16-, and17-positions, 3-, 4-, 16-, and 17-positions or the 3-, 11-, 16-, and17-positions. The hydroxyl, esters or ethers respectively can be in the2- and/or 3-β,β,β,β-17, 2- and/or 3-β,β,β,α-17, 2- and/or 3-β,β,α,β-17,2- and/or 3-β,α,β,β-17, 2- and/or 3-α,β,β,β-17, 2- and/or 3-β,β,α,α-17,2- and/or 3-β,α,β,α-17, or in the 2- and/or 3-α,β,β,α-17 configurationswhen no double bond is present at the 4-position. The term “2- and/or3-β,β,β,β-17” means that the 2-position is optionally substituted andthe 3- and 17-positions are substituted with hydroxyl, ester or ether.The hydroxyl, esters or ethers respectively can be in the 2- and/or3-β,α,α,β-17, 2- and/or 3-α,β,α,β-17, 2- and/or 3-α,α,β,β-17, 2- and/or3-β,α,α,α-17, 2- and/or 3-α,β,α,α-17, 2- and/or 3-α,α,β,α-17, 2- and/or3-α,α,α,β-17, 2- and/or 3-β,α,α,α-17, configurations when no double bondis present at the 4-position. R¹⁰ in these compounds is typically —H or—F and R⁵ optionally is —CH₃ or —C₂H₅ and R⁶ optionally is —H, —CH₃,—CH₂OH, —CCH or —CCCH₃. For these compounds, the one, two or more of 2-,3-, 4-, 7-, 11-, 16- and 17-positions are substituted with —H oroptionally substituted alkyl such as —CH₃, —C₂H₅, —CH₂═CH₂, —CCH, —CF₃or —C₂F₅. Exemplary compounds include17α-ethynylandrost-5-ene-2β,3β,7β,17β-tetrol,17α-chloroethynylandrost-5-ene-2β,3β,7β,17β-tetrol,17α-ethynylandrost-5-ene-2β,3α,7β,17β-tetrol,17α-chloroethynylandrost-5-ene-2β,3α,7β,17β-tetrol,3β-acetoxy-17α-ethynylandrost-5-ene-2β,7β,17β-triol,7β-acetoxy-17α-ethynylandrost-5-ene-2α,3β,17β-triol and3β,17β-diacetoxy-17α-ethynylandrost-5-ene-2β,7β-diol. Other exemplarycompounds include 17α-ethynylandrost-5-ene-2α,3β,11β,17β-tetrol,17α-chloroethynylandrost-5-ene-2α,3β,11β,17β-tetrol,17α-ethynylandrost-5-ene-2β,3α,11β,17β-tetrol,3β-acetoxy-17α-ethynylandrost-5-ene-2α,11β,17β-triol,3α-acetoxy-17α-ethynylandrost-5-ene-2α,11α,17β-triol,3β,17β-diacetoxy-17α-ethynylandrost-5-ene-2α,11β-diol,17α-ethynylandrost-5-ene-2α,3β,16α,17β-tetrol,17α-chloroethynylandrost-5-ene-2α,3β,16α,17β-tetrol,17α-ethynylandrost-5-ene-2β,3α,16β,17β-tetrol,3β-acetoxy-17α-ethynylandrost-5-ene-2α,16α,17β-triol,3α-acetoxy-17α-ethynylandrost-5-ene-2α,16α,17β-triol and3β,17β-diacetoxy-17α-ethynylandrost-5-ene-2α,16α-diol.

In some embodiments of the formula 1 compounds, five independentlyselected hydroxyl, ester and/or ether moieties can be present. For thesecompounds, hydroxyl, ester and/or ether moieties are usually present atthe 3- and 17-positions and at 3 other positions. These substituents canbe at the 2-, 3-, 4-, 7- 11-, 17- and 18-positions, e.g., at the 2-, 3-,7- 16- and 17-positions or 2-, 3-, 11- 16- and 17-positions.Independently selected hydroxyl, ester and/or ether moieties can bepresent at the 3-, 4-, 7- 11- and 17-positions, 3-, 4-, 11- 16- and17-positions, 3-, 4-, 7- 16- and 17-positions or the 3-, 7-, 11-, 16-,17-positions. For these compounds, each moiety can be in theα-configuration or the β-configuration when no double bond is present atthe 4-position. Thus, substituents in these compounds can respectivelybe in the 2- and/or 3-β,β,β,β,β-17, 2- and/or 3-β,β,β,β,α-17, 2- and/or3-β,β,β,α,β-17, 2- and/or 3-β,β,α,β,β-17, 2- and/or 3-β,α,β,β,β-17, 2-and/or 3-αβ,β,β,β-17, 2- and/or 3-β,β,β,αα-17, 2- and/or 3-β,β,α,β,α-17,2- and/or 3-β,α,β,β,α-17, 2- and/or 3-α,β,β,β,α-17, 2- and/or3-β,β,α,α,β-17, 2- and/or 3-β,α,β,α,β-17, 2- and/or 3-α,β,β,α,β-17, 2-and/or 3-β,α,α,β,β-17, 2- and/or 3-α,β,α,β,β-17,2- and/or 3-α,α,β,β,β-17or in the 2- and/or 3-β,β,α,α,α-17 configurations respectively. Thesubstituents in these compounds can also respectively be in the 2-and/or 3-β,α,β,α,α-17, 2- and/or 3-β,α,α,β,α-17, 2- and/or3-β,α,α,α,β-17, 2- and/or 3-α,β,β,α,α-17, 2- and/or 3-α,β,α,β,α-17, 2-and/or 3-β,α,α,α,β-17, 2- and/or 3-αα,β,β,α-17, 2- and/or3-α,α,β,α,β-17, 2- and/or 3-α,α,α,β,β-17, 2- and/or 3-β,α,α,α,α-17, 2-and/or 3-α,β,α,α,α17, 2- and/or 3-α,α,β,α,α-17, 2- and/or3-α,α,α,β,α-17, 2- and/or 3-α,α,α,α,β-17 or in the 2- and/or3-α,α,α,α,α-17 configurations respectively. Exemplary compounds include(1) 17α-ethynylandrost-5-ene-2β,3β,7β,16α,17β-pentol,17α-chloroethynylandrost-5-ene-2α,3β,7β,16α,17β-pentol,17α-ethynylandrost-5-ene-2β,3α,7β,16α,17β-pentol,17α-chloroethynylandrost-5-ene-2β,3α,7β,16α,17β-pentol,3β-acetoxy-17α-ethynylandrost-5-ene-2β,7β,16α,17β-tetrol,17β-acetoxy-17α-ethynylandrost-5-ene-2β,3α,7β,16α-tetrol,3β,2α-diacetoxy-17α-ethynylandrost-5-ene-7β,16α,17β-triol and isomers ofany of these compounds where the moiety at the 3-position or the7-position is inverted, e.g., 3β-OH or 3β-acetate is inverted to 3α-OH3α-acetate, (2) 17α-ethynylandrost-5-ene-3β,4α,7β,17β,18-pentol,17α-chloroethynylandrost-5-ene-3α,4β,7α,17β,18-pentol,17α-ethynylandrost-5-ene-3β,4α,7α,17β,18-pentol,3β-acetoxy-17α-ethynylandrost-5-ene-4α,7α,17β,18-tetrol and isomers ofany of these compounds where the moiety at the 3-position or the7-position is inverted and (3)17α-ethynylandrost-5-ene-3β,4α,16α,17β,18-pentol,17α-ethynylandrost-5-ene-3β,4α,16β,17β,18-pentol,17α-chloroethynylandrost-5-ene-3β,4α,16α,17β,18-pentol,3β-acetoxy-17α-ethynylandrost-5-ene-4α,16α,17β,18-tetrol,4β-acetoxy-17α-ethynylandrost-5-ene-3α,16α,17β,18-tetrol,3β,16α-diacetoxy-17α-ethynylandrost-5-ene-4α,17β,18-triol and isomers ofany of these compounds where the moiety at the 3-position or the4-position, if present, is inverted.

For the formula 1 compounds described herein, the 16-position may beunsubstituted, i.e., one or both R³ are —H, and a second R⁴ can bepresent at the 17-position in the α-configuration or the β-configurationwhen no double bond is present at the 17-position. Such second R⁴moieties include C₁₋₈ optionally substituted alkyl such as —CH₃, —C₂H₅,—CF₃, —C₂F₅, —C═CH₂, —CCH, —CCCl—CCCH₃ or —CCCH₂Cl.

For any of these compounds, the 2-position may be unsubstituted, i.e.,R⁹ is —CH₂—. In some embodiments, R⁹ is substituted, e.g., —O—,—CH(OH)—, —CH(ester)- or —CH(ether)- where the hydroxyl, ester or ethermoiety is in the α- or β-configuration. Exemplary R⁹ moieties are—CH(α-OH)—, —CH(β-OH)—, —C(β-CH₃)(α-OH)—, —C(α-CH₃)(β-OH)—,—CH(α-OCH₃)—, —CH(β-OCH₃)—, —CH(α-OC(O)CH₃)— and —CH(β-OC(O)CH₃)—. OtherR⁹ moieties are —CH(α-OC(O)CH₂CH₃)—, —CH(β-OC(O)CH₂CH₃)—,—CH(α-OCH₂CH₃)—, —CH(β-OCH₂CH₃)—, —C(O—CH₃)(α-OC(O)CH₃)— and—C(α-CH₃)(β-OC(O)CH₃)—.

Exemplary F1Cs that can be used to treat metabolic diseases, lungconditions, autoimmune, inflammatory or other conditions describedherein. These compounds include4β-fluoro-17α-ethynylandrost-5-ene-3β,7β, 17β-triol,4α-fluoro-17α-ethynylandrost-5-ene-3β,7β,17β-triol,4β-fluoro-17α-ethynylandrost-5-ene-3α,7β,17β-triol,4α-fluoro-17α-ethynylandrost-5-ene-3α,7β,17β-triol,4β-fluoro-17α-ethynylandrost-5-ene-3β,7α,17β-triol,4α-fluoro-17α-ethynylandrost-5-ene-3β,7β,17β-triol,4β-fluoro-17α-ethynylandrost-5-ene-3α,7α,17β-triol,4α-fluoro-17α-ethynylandrost-5-ene-3α,7α,17β-triol,17α-ethynylandrost-5-ene-3β,7β,17β-triol,17β-ethynylandrost-5-ene-3β,7β,17α-triol,17α-ethynylandrost-5-ene-3β,7β-diol-7-one,17α-ethynylandrost-5-ene-3α,17β-diol-7-one,17α-ethynylandrost-5-ene-3β,7β,17β-triol,17α-ethynylandrost-5-ene-3β,7α,17β-triol and an analog of any of thesecompounds where (1) an ester such as acetate replaces the hydroxyl groupat the 3-position or at the 7-position, e.g.,7β-acetoxy-17α-ethynylandrost-5-ene-3β,17β-diol,7β-acetoxy-17α-ethynylandrost-5-ene-3α,17β-diol,3β-acetoxy-4β-fluoro-17α-ethynylandrost-5-ene-7β,17β-diol or7β-acetoxy-4β-fluoro-17α-ethynylandrost-5-ene-3α,17β-diol or (2) theethynyl moiety at the 17-position is replaced with an optionallysubstituted C₂₋₄ alkynyl moiety such as chloroethynyl. Other F1Csinclude 17α-ethynylandrost-5-ene-3β,4α,7β,17β-tetrol,17α-ethynylandrost-5-ene-3α,4α,7β,17β-tetrol,17α-ethynylandrost-5-ene-3β,4β,7β,17β-tetrol,17α-ethynylandrost-5-ene-3α,4β,7β,17β-tetrol,17α-ethynylandrost-5-ene-3β,4α,7α,17β-tetrol and17α-ethynylandrost-5-ene-3α,4α,7α,17β-tetrol. Other F1C analogs of theforegoing compounds have an optionally substituted C2-4 alkynyl moietysuch as —C≡C—Cl, —CCCH₃ or —C≡C—CH₂Cl that replaces the ethynyl moietyat the 17-position, e.g.,4α-fluoro-17α-chloroethynylandrost-5-ene-3β,7β,17β-triol,4α-fluoro-17α-chloroethynylandrost-5-ene-3α,7β,17β-triol,17α-chloroethynylandrost-5-ene-3β,7β,17β-triol,17α-chloroethynylandrost-5-ene-3β,7α,17β-triol and17α-chloroethynylandrost-5-ene-3α,7β,17β-triol.

F1Cs also include 4β-fluoro-17α-ethynylandrostane-3β,7β,17β-triol,4α-fluoro-17α-ethynylandrostane-3β,7β,17β-triol,4β-fluoro-17α-ethynylandrostane-3α,7β,17β-triol,4α-fluoro-17α-ethynylandrostane-3α,7β,17β-triol,4β-fluoro-17α-ethynylandrostane-3β,7α,17β-triol,4α-fluoro-17α-ethynylandrostane-3β,7α,17β-triol,4β-fluoro-17α-ethynylandrostane-3α,7α,17β-triol,4α-fluoro-17α-ethynylandrostane-3α,7α,17β-triol,17α-ethynylandrostane-3β,7β,17β-triol,17α-ethynylandrostane-3β,17β-diol-7-one,17α-ethynylandrostane-7β,17β-diol-3-one,17α-ethynylandrostane-3α,7β,17β-triol,17α-ethynylandrostane-3β,7α,17β-triol and an analog of any of thesecompounds wherein an ester such as acetate replaces the hydroxyl groupat the 3-position or at the 7-position, e.g.,7β-acetoxy-17α-ethynylandrostane-3β,17β-diol,7β-acetoxy-17α-ethynylandrostane-3α,17β-diol,3β-acetoxy-4β-fluoro-17α-ethynylandrostane-7β,17β-diol,7β-acetoxy-4β-fluoro-17α-ethynylandrostane-3α,17β-diol,17α-ethynylandrostane-3β,4α,7β,17β-tetrol,17α-ethynylandrostane-3α,4α,7β,17β-tetrol,17α-ethynylandrostane-3β,4β,7β,17β-tetrol,17α-ethynylandrostane-3α,4α,7β,17β-tetrol, 17α-ethynylandrostane-3β,4α,7α,17β-tetrol and 17α-ethynylandrostane-3α, 4α, 7α,17β-tetrol. Other F1Canalogs of the foregoing compounds have an optionally substituted C2-4alkynyl moiety such as —C≡C—Cl, —CCCH₃ or —C≡C—CH₂Cl that replaces theethynyl moiety at the 17-position, e.g.,4α-fluoro-17α-chloroethynylandrostane-3β, 7β,17β-triol,4α-fluoro-17α-chloroethynylandrostane-3α,7β,17β-triol,17α-chloroethynylandrostane-3β,7β,17β-triol,17α-chloroethynylandrostane-3β,7α,17β-triol and17α-chloroethynylandrostane-3α,7β,17β-triol.

F1Cs include 4β-fluoro-17α-ethynylandrost-4-ene-3β,7β,17β-triol,4α-fluoro-17α-ethynylandrost-4-ene-3β,7β,17β-triol,4β-fluoro-17α-ethynylandrost-4-ene-3α,7β,17β-triol,4α-fluoro-17α-ethynylandrost-4-ene-3α,7β,17β-triol,4β-fluoro-17α-ethynylandrost-4-ene-3β,7α,17β-triol,4α-fluoro-17α-ethynylandrost-4-ene-3β,7α,17β-triol,4β-fluoro-17α-ethynylandrost-4-ene-3α,7α,17β-triol,4α-fluoro-17α-ethynylandrost-4-ene-3α,7α,17β-triol,17α-ethynylandrost-4-ene-3β,7β,17β-triol,17α-ethynylandrost-4-ene-3β,7β,17β-triol,17α-ethynylandrost-4-ene-3β,7α,17β-triol,17α-ethynylandrost-4-ene-3β,17β-diol-7-one,17α-ethynylandrost-4-ene-7β,17β-diol-3-one,17α-ethynylandrost-4-ene-3α,17β-diol-7-one and an analog of any of thesecompounds wherein an ester such as acetate replaces the hydroxyl groupat the 3-position or at the 7-position, e.g.,7β-acetoxy-17α-ethynylandrost-4-ene-3β,17β-diol,7β-acetoxy-17α-ethynylandrost-4-ene-3α,17β-diol,3β-acetoxy-4β-fluoro-17α-ethynylandrost-4-ene-7β,17β-diol,7β-acetoxy-4β-fluoro-17α-ethynylandrost-4-ene-3α,17β-diol,17α-ethynylandrost-4-ene-3β,4α,7β,17β-tetrol,17α-ethynylandrost-4-ene-3α,4α,7β,17β-tetrol,17α-ethynylandrost-4-ene-3β,4β,7β,17β-tetrol,17α-ethynylandrost-4-ene-3α,4β,7β,17β-tetrol,17α-ethynylandrost-4-ene-3β,4α,7α,17β-tetrol and17α-ethynylandrost-4-ene-3α,4α,7α,17β-tetrol. Other F1C analogs of theforegoing compounds have an optionally substituted C₂₋₄ alkynyl moietysuch as —C≡C—Cl, —CCCH₃ or —C≡C—CH₂Cl that replaces the ethynyl moietyat the 17-position, e.g.,4α-fluoro-17α-chloroethynylandrost-4-ene-3β,7β,17β-triol,4α-fluoro-17α-chloroethynylandrost-4-ene-3α,7β,17β-triol,17α-chloroethynylandrost-4-ene-3β,7β,17β-triol,17α-chloroethynylandrost-4-ene-3β,7α,17β-triol and17α-chloroethynylandrost-4-ene-3α,7β,17β-triol.

Other exemplary F1Cs include4β-fluoro-17α-ethynylandrost-5-ene-3β,16α,17β-triol,4α-fluoro-17α-ethynylandrost-5-ene-3β,16α,17β-triol,4β-fluoro-17α-ethynylandrost-5-ene-3α,16α,17β-triol,4α-fluoro-17α-ethynylandrost-5-ene-3α,16α,17β-triol,4β-fluoro-17α-ethynylandrost-5-ene-3β,16β,17β-triol,4α-fluoro-17α-ethynylandrost-5-ene-3β,16β,17β-triol,4β-fluoro-17α-ethynylandrost-5-ene-3α,16α,17β-triol,4α-fluoro-17α-ethynylandrost-5-ene-3α,16α,17β-triol,17α-ethynylandrost-5-ene-3β,4α,16α,17β-tetrol,17α-ethynylandrost-5-ene-3α, 4α,16α,17β-tetrol,17α-ethynylandrost-5-ene-3β,4β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3α, 4β,16α,17β-tetrol,17α-ethynylandrost-5-ene-4α,16α,17β-triol-3-one, and an analog of any ofthese compounds wherein an ester such as acetate replaces the hydroxylgroup at the 3-position or at the 16-position, e.g.,16α-acetoxy-17α-ethynylandrost-5-ene-3β,17β-diol,16α-acetoxy-17α-ethynylandrost-5-ene-3α,17β-diol,3β-acetoxy-4β-fluoro-17α-ethynylandrost-5-ene-16α,17β-diol,16β-acetoxy-4β-fluoro-17α-ethynylandrost-5-ene-3α,17β-diol,3β-acetoxy-17α-ethynylandrost-5-ene-4β,16α,17β-triol,3α-acetoxy-17α-ethynylandrost-5-ene-4β,16α,17β-triol,16α-acetoxy-17α-ethynylandrost-5-ene-3β,4β,17β-triol and16α-acetoxy-17α-ethynylandrost-5-ene-3α,4β,17β-triol. Other F1C analogsof the foregoing compounds have an optionally substituted C2-4 alkynylmoiety such as —C≡C—Cl, —CCCH₃ or —C≡C—CH₂Cl that replaces the ethynylmoiety at the 17-position, e.g.,4β-fluoro-17α-chloroethynylandrost-5-ene-3β,16α,17β-triol,4α-fluoro-17α-chloroethynylandrost-5-ene-3β,16α,17β-triol,17α-chloroethynylandrost-5-ene-3β,4α,16α,17β-tetrol and17α-chloroethynylandrost-5-ene-3α,4α,16α,17β-tetrol.

Other exemplary F1Cs include4β-fluoro-17α-ethynylandrost-4-ene-3β,16α,17β-triol,4α-fluoro-17α-ethynylandrost-4-ene-3β,16α,17β-triol,4β-fluoro-17α-ethynylandrost-4-ene-3α,16α,17β-triol,4α-fluoro-17α-ethynylandrost-4-ene-3α,16α,17β-triol,4β-fluoro-17α-ethynylandrost-4-ene-3β,16β,17β-triol,4α-fluoro-17α-ethynylandrost-4-ene-3β,16β,17β-triol,4β-fluoro-17α-ethynylandrost-4-ene-3α,16α,17β-triol,4α-fluoro-17α-ethynylandrost-4-ene-3α,16α,17β-triol,17α-ethynylandrost-4-ene-3β,4α,16α,17β-tetrol,17α-ethynylandrost-4-ene-3α,4α,16α,17β-tetrol,17α-ethynylandrost-4-ene-3β,4β,16α,17β-tetrol,17α-ethynylandrost-4-ene-3α,4β,16α,17β-tetrol,4β-fluoro-17α-ethynylandrost-4-ene-16α,17β-diol-3-one,17α-ethynylandrost-4-ene-4α,16α,17β-triol-3-one and an analog of any ofthese compounds wherein an ester such as acetate replaces the hydroxylgroup at the 3-position or at the 16-position, e.g.,16α-acetoxy-17α-ethynylandrost-4-ene-3β,17β-diol,16α-acetoxy-17α-ethynylandrost-4-ene-3α,17β-diol,3β-acetoxy-4β-fluoro-17α-ethynylandrost-4-ene-16α,17β-diol,16β-acetoxy-4β-fluoro-17α-ethynylandrost-4-ene-3α,17β-diol,3β-acetoxy-17α-ethynylandrost-4-ene-4β,16α,17β-tetrol,3α-acetoxy-17α-ethynylandrost-4-ene-4β,16α,17β-tetrol,16α-acetoxy-17α-ethynylandrost-4-ene-3β,4β,17β-triol and16α-acetoxy-17α-ethynylandrost-4-ene-3α,4β,17β-triol. F1C analogs of theforegoing compounds have an optionally substituted C2-4 alkynyl moietysuch as —C≡C—Cl, —CCCH₃ or —C≡C—CH₂Cl that replaces the ethynyl moietyat the 17-position, e.g.,4β-fluoro-17α-chloroethynylandrost-4-ene-3α,16α,17β-triol,4α-fluoro-17α-chloroethynylandrost-4-ene-3β,16α,17β-triol,17α-chloroethynylandrost-4-ene-3β,4α,16α,17β-tetrol and17α-chloroethynylandrost-4-ene-3α, 4α,16α,17β-tetrol.

Other exemplary F1Cs include4β-fluoro-17α-ethynylandrostane-3β,16α,17β-triol,4α-fluoro-17α-ethynylandrostane-3β,16α,17β-triol,4β-fluoro-17α-ethynylandrostane-3α,16α,17β-triol,4α-fluoro-17α-ethynylandrostane-3α,16α,17β-triol,4β-fluoro-17α-ethynylandrostane-3β,16β,17β-triol,4α-fluoro-17α-ethynylandrostane-3β,16β,17β-triol,4β-fluoro-17α-ethynylandrostane-3α,16α,17β-triol,4α-fluoro-17α-ethynylandrostane-3α,16α,17β-triol,17α-ethynylandrostane-3β,4α,16α,17β-tetrol, 17α-ethynylandrostane-3α,4α,16α,17β-tetrol, 17α-ethynylandrostane-3β,4β,16α,17β-tetrol,17α-ethynylandrostane-3α,4β,16α,17β-tetrol,4β-fluoro-17α-ethynylandrostane-16α,17β-diol-3-one,17α-ethynylandrostane-4β,16α,17β-triol-3-one, and an analog of any ofthese compounds wherein an ester such as acetate replaces the hydroxylgroup at the 3-position (if present), 4-position or at the 16-position,e.g., 16α-acetoxy-17α-ethynylandrostane-3β,4α,17β-triol,16α-acetoxy-17α-ethynylandrostane-3β,4β,17β-triol,16α-acetoxy-17α-ethynylandrostane-3α,4α,17β-triol,4α-acetoxy-17α-ethynylandrostane-3β,16α,17β-triol,16β-acetoxy-4β-fluoro-17α-ethynylandrostane-3α, 17β-diol,3β-acetoxy-17α-ethynylandrostane-4β,16α,17β-tetrol,3α-acetoxy-17α-ethynylandrostane-4β,16α,17β-tetrol,16α-acetoxy-17α-ethynylandrostane-3β,4β,17β-triol and16α-acetoxy-17α-ethynylandrostane-3α,4β,17β-triol. F1C analogs of theforegoing compounds have an optionally substituted C2-4 alkynyl moietysuch as —C≡C—Cl, —CCCH₃ or —C≡C—CH₂Cl that replaces the ethynyl moietyat the 17-position, e.g.,4α-fluoro-17α-chloroethynylandrostane-3β,16α,17β-triol,4α-fluoro-17α-chloroethynylandrostane-3α,16α,17β-triol,17α-chloroethynylandrostane-3β,4α,16α,17β-tetrol and17α-chloroethynylandrostane-3α,4α,16α,17β-tetrol.

Biodynamic compounds. The method to identify or characterize abiodynamic compound comprising can be accomplished as described above.The method optionally further comprises conducting a protocol todetermine if the test compound modulates the activity or level of themediator of the acute biological response by about 20% or about 25% toabout 70% or about 75% in an assay in vitro, optionally wherein the testcompound does not activate or antagonize a glucocorticoid receptor bymore than about 10%, about 20% or about 30% when compared to a suitablereference activator or antagonist of the glucocorticoid receptor, e.g.,dexamethasone or cortisol. In these embodiments, the acute stimulus orbiological insult can be exposure of the subject to a sufficient amountof ionizing radiation or a proinflammatory signal, compound orcomposition, optionally wherein the proinflammatory signal, compound orcomposition is bacterial LPS or TNFα, and/or optionally wherein themediator of the acute biological response is NF-κB or IκB.

The acute stimulus or biological insult can be administration ofsufficient bacterial LPS to a sufficient number of drug treated mice anda sufficient number vehicle control mice and measurement of the effectof the test compound on the mediator of the acute biological response ata time when (i) the acute response is maximal or nearly maximal,optionally at about 1.5 hours, e.g., at about 70-110 minutes or 75-105minutes, after administration of bacterial LPS by intraperitonealinjection and (ii) one or two other time points before and/or after theadministration of the sufficient bacterial LPS, optionally at one timepoint before the administration of the sufficient bacterial LPS and atone later time after the acute response is maximal or nearly maximal,optionally at about 2.0 or 2.5 hours after administration of bacterialLPS by intraperitoneal injection, and optionally wherein the mediator ofthe acute biological response is NF-κB or IκB.

The administration of sufficient bacterial LPS can optionally beaccomplished essentially according to the methods described herein or asuitable variation thereof and optionally wherein the capacity of thecompound to partially modulate the level or activity of the mediator ofthe acute biological response is accomplished essentially according to amethod described herein or a suitable variation thereof.

Other stimuli or biological insults that can be analyzed includeischemia and reperfusion of one or more ischemic tissues, thermal orchemical burns of relatively low, moderate or high severity.

The capacity of 17α-ethynylandrost-5-ene-3β,7β,17β-triol to exert atransient, but very potent, effect, which fades, and normal functionreturns is referred to herein as a biodynamic response (see example 9).A biodynamic response elicited by a ‘biodynamic agent’ such as17α-ethynylandrost-5-ene-3β,7β,17β-triol contrasts with a ‘biostaticresponse’ that a compound such as dexamethasone elicits toward itseffector biomolecules such as the glucocorticoid receptor or NF-κB,which it inhibits indirectly through activation of the glucocorticoidreceptor. The biostatic response essentially is an ‘all on all the time’response with the biological potency of a ‘biostatic agent’ such asdexamethasone having relatively little variation at a givenconcentration at target cells or tissues.

The pharmacodynamic effect of a biostatic agent appears to varyprimarily with its concentration or pharmacokinetic properties. Bycontrast, biodynamic agents such as17α-ethynylandrost-5-ene-3β,7β,17β-triol are characterized by apharmacodynamic effect that is affected by a combination of itsconcentration at target cells or tissues and the nature and intensity ofthe underlying biological stimulus. Thus, a biological stimuluselicited, e.g., by exposure to a potentially lethal amount of ionizingradiation such a γ-rays or X-rays or exposure to bacterial LPS, TNFα oranother agent that can activate or inhibit mediators of inflammationsuch as NF-κB, IκB, IL-6, C reactive protein. In this regard, biodynamicdrugs can exhibit a looser statistical correlation, or no significantcorrelation, between pharmacokinetic and pharmacodynamic effectscompared to what is generally observed for biostatic drugs.

One aspect of biodynamic drugs is their potential capacity to decreasesystemic toxicity associated with biostatic drugs that may act at leastin part through modulating the same or similar target biomolecules.Biostatic dugs such as dexamethasone can be used clinically to treat awide range of inflammation conditions, but the ‘all on all the time’bioactivity can lead to toxicity. In the case of inhibiting NF-κB,constant and relatively complete inhibition of its activity, e.g.,inhibition by about 75%, 80%, 85%, 90%, 95% or essentially 100% in mostor all tissues, for a prolonged time, e.g., for more than 1, 2 or 4hours to about 1, 2, 3 days or more, can result in observable unwantedside-effects since some basal level of NF-κB activity is needed fornormal biological function in most tissues. Known toxicities associatedwith the use of glucocorticoids such as dexamethasone are likely toarise at least in part from the relatively complete shut-down ofaffected biomolecules such as NF-κB. By contrast, biodynamic drugs canexert a more transient response that can lead to an amelioration ordecrease in observable toxic side effects.

Another aspect of biodynamic drugs is their capacity to potentiallyexert a therapeutic effect in a tissue-specific manner. Thus, ananimal's response to a challenge such as exposure to a biological insultsuch as a potentially lethal amount of bacterial LPS or reperfusion ofaffected tissues after transient ischemia may be manifested by varyingdegrees of NF-κB activation in varying tissues. A biodynamic drug couldact in tissues where the animal's response is relatively great, e.g.,mouse spleen or cardiac tissue, while leaving the function of NF-κBrelatively unaffected in other tissues, e.g., brain, where the responseto the biological insult is relatively lower for the target biomoleculethat at least partially mediates the response to the biological insult.

Biodynamic drugs may act in part by their capacity to partially inhibittarget biomolecules. As described below in example 7,17α-ethynylandrost-5-ene-3β,7β,17β-triol and some other compoundsdescribed there partially inhibited activation of NF-kB in the cells invitro, but complete inhibition was not observed at any concentration.For most of these compounds the degree of inhibition of NF-kB was about25-80%, typically about 30-65% or about 30-70%, an unexpected phenomenonthat was not previously described for these compounds. This contrastedwith the activity of the biostatic drug dexamethasone, which completelyinhibited NF-κB activity at a sufficiently high concentration. Thispartial inhibition of NF-κB in vitro appears to be partially reflectedby its activity described in this example. Thus, in at least some cases,biodynamic drugs are characterized by having a capacity to partiallyinhibit or activate a target biomolecule in a system such as the invitro assay described below. The inhibition of NF-κB appears to beindirect, since it is not believed at present that17α-ethynylandrost-5-ene-3β,7β,17β-triol and the other compoundsdescribed here bind directly to NF-κB in the cytoplasm or the nucleus.

The protocol described in example 7 below, or suitable variations of it,can be used to characterize other compounds for their potential to actas biodynamic or biostatic agents by modulating (detectably activatingor detectably antagonizing or inhibiting) molecules such as NF-κB invivo, where such modulation is typically partial inhibition or partialactivation. The compounds can also be analyzed in in vitro assays suchas the assay described in example 7 to further characterize theirmechanism of action. Suitable variations of the in vivo protocolsdescribed herein include using various dosages of test compounds andvarious routes of administration, e.g., a dose range of about 0.1 mg/kgto about 350 mg/kg administered orally, buccally, sublingually orparenterally such as intradermal, subcutaneous, intravenous orintramuscular injection or by intranasal or inhalation to the nasalpassages, vomeronasal organ or lung alveoli or airway passages leadingto the alveoli, e.g., bronchi or bronchioli. Suitable test dosages willtypically be about 1-150 mg/kg. Biomolecules that can be measured invivo such as IκB, kinases such as src kinase, a map kinase or othersignal mediators described herein. Such characterization methods can beconducted in one or more of a range of subjects such as rodents, e.g.,rats, dogs, non-human primates such as rhesus or cynomolgus monkeys.Groups of animals consisting of about 3-12 animals per group, e.g., 4-8animals per group, can be used with suitable vehicle or placebocontrols, test compounds that are potential biodynamic drugs, positivebiodynamic drug controls such as17α-ethynylandrost-5-ene-3β,7β,17β-triol, positive biostatic drugcontrols such as dexamethasone and groups where the response of the testcompound in different cell populations or tissues are compared againstone or more control groups, e.g., vehicle controls, positive or negativebiodynamic drug controls or positive or negative biostatic drugcontrols. Other compounds that can be used in these methods as controlor reference compounds include 17α-ethynylandrost-5-ene-3α,7β,17β-triol,17α-ethynylandrost-5-ene-3β,7α,17β-triol, 17α-ethynylandrost-5-ene-3β,17β-diol-7-one, 17α-ethynylandrost-5-ene-3β,7β,16α,17β-tetrol and17α-ethynylandrost-5-ene-3α,7β,16α,17β-tetrol.

When such analyses are applied to humans, the range of experimentaloptions will naturally differ compared to other animals. Human tissuesor samples such as blood, bone marrow or lung lavage fluid will be morereadily accessible for analyses than other tissue types such as spleenor liver, which need to be obtained by invasive techniques. Thus, ingeneral, animal studies will be conducted before human responseassessment.

Inflammation treatments. An aspect of some claimed embodiments is thatthe formula 1 compounds can decrease inflammation by affecting mediatorsof inflammation such as NF-κB, IL-6 or TNFα. The NF-κB molecule often isan important mediator of inflammation. Increased activation of NF-κB isassociated with a range of inflammatory diseases and autoimmuneconditions. Anti-inflammatory activity from compounds in vivo couldarise, e.g., from eliciting prostaglandin synthesis and other activityin liver, leading to a systemic anti-inflammation response.Alternatively, anti-inflammation activity for compounds could arise fromthe capacity of the compounds to inhibit stimulation of NF-κB activitythat arises from sources other than LPS. A number of different materialscan activate NF-κB activity, including LPS, TNF-α, IL-1, the presence ofcertain viral or bacterial gene products, activation of B-cells orT-cells, or exposure of cells to ultraviolet radiation. Not all celltypes can respond to all of these stimuli since not all cells expressthe signaling machinery that is needed to respond to each of thesestimuli. Most cell types can respond to one or a few of these signals,but rarely can a given cell type respond to all.

The formula 1 compounds can be used to treat or ameliorate conditions orsymptoms associated with conditions. Conditions and symptoms includeinflammation such as pain, fever or fatigue; endometriosis; fever;fibromyalgia; a myelitis condition such as acute transverse myelitis;glomerulonephritis; graft versus host disease, organ or tissuetransplant rejection, e.g., kidney, lung, bone marrow or livertransplant; hemorrhagic shock; fibromyalgia; hyperalgesia; inflammatorybowel disease; gastritis; irritable bowel syndrome; ulcerative colitis;a peptic ulcer; a stress ulcer; a bleeding ulcer; gastric hyperacidity;dyspepsia; gastroparesis; gastroesophageal reflux disease; inflammatoryconditions of a joint, including osteoarthritis, psoriatic arthritis andrheumatoid arthritis; inflammatory eye disease, as may be associatedwith, e.g., corneal transplant; ischemia, including cerebral ischemia(e.g., brain injury as a result of trauma, epilepsy, hemorrhage orstroke, each of which may lead to neurodegeneration); Kawasaki'sdisease; learning impairment; lung diseases (e.g., ARDS); ademyelinating condition such as multiple sclerosis or progressivemultifocal leukoencephalopathy, which may be remitting or relapsing;myopathies (e.g., muscle protein metabolism, especially in sepsis);neurotoxicity (e.g., as induced by HIV); osteoporosis; pain, includingcancer-related pain; Parkinson's disease; Alzheimer's disease;periodontal disease; pre-term labor; psoriasis; reperfusion injury;septic shock; side effects from radiation therapy; temporal mandibularjoint disease; alcohol-induced liver injury including alcoholiccirrhosis; rheumatic fever; sarcoidosis; scleroderma; chronic fatiguesyndrome; coronary conditions and indications, including congestiveheart failure, coronary restenosis, myocardial infarction, myocardialdysfunction (e.g., related to sepsis), and coronary artery bypass graft;sleep disturbance; uveitis; seronegative polyarthritis; ankylosingspondylitis; Reiter's syndrome and reactive arthritis; Still's disease;psoriatic arthritis; enteropathic arthritis; polymyositis;dermatomyositis; scleroderma; systemic sclerosis; vasculitis (e.g.,Kawasaki's disease); inflammation resulting from, e.g., strain, sprainor cartilage damage; wound healing; thin or fragile skin; petechiae orecchymoses; erythema; and trauma. Trauma includes wounds, chemicalburns, thermal burns, radiation burns and tissue or organ damageassociated with a surgery such as an orthopedic surgery or an abdominalsurgery. Inflammation conditions can include inflammation associatedwith reperfusion injury, restenosis after angioplasty, myocardial orcerebral infarction.

Unwanted inflammation conditions or symptoms, include lung inflammationconditions, e.g., cystic fibrosis, acute asthma, chronic asthma, steroidresistant asthma, acute bronchitis, chronic bronchitis, emphysema,psoriasis, eczema, adult respiratory distress syndrome (ARDS) or chronicobstructive pulmonary disease (COPD).

Autoimmune and pulmonary conditions. In some claimed embodiments, theformula 1 compounds (F1Cs) or compositions described herein can be usedto treat, prevent or slow the progression of autoimmune or relatedconditions such as type 1 diabetes, Crohn's disease, arthritis, contactdermatitis, lupus and multiple sclerosis (MS) conditions. MS conditionsinclude relapsing-remitting MS and secondary progressive MS. The lupusconditions include systemic lupus erythematosus, lupuserythematosus-related arthritis, lupus erythematosus-related skinchanges, lupus erythematosus-related hematologic abnormalities, lupuserythematosus-related kidney impairment, lupus erythematosus-relatedheart or lung disease, lupus erythematosus-related neuropsychiatricchanges, lupus erythematosus-related tissue inflammation, discoid lupuserythematosus, subacute cutaneous lupus erythematosus and drug-inducedlupus erythematosus. Arthritis and related conditions include rheumatoidarthritis, osteoarthritis, fibromyalgia, primary osteoarthritis,secondary osteoarthritis, psoriatic arthritis, lupuserythematosus-related arthritis, arthritis associated with acute orchronic inflammatory bowel disease or colitis, arthritis associated withankylosing spondylitis, arthritis-related tissue inflammation, jointpain, joint stiffness, impaired joint movement, joint swelling, jointinflammation and synovium inflammation.

In these claimed embodiments, the F1Cs or compositions containing a F1Cand one or more excipients can be used to treat, prevent, delay theonset of or slow the progression of conditions such as ankylosingspondylitis, psoriasis, eczema, a dermatitis such as contact dermatitis,a colitis such as ulcerative colitis, Crohn's disease, acute or chronicinflammatory bowel disease, autoimmune renal injury and liver injury.

Experimental autoimmune encephalomyelitis (EAE) is an experimentalcondition in animals that has clinical, histopathological andimmunological characteristics similar to human MS and, as with MS,exhibits infiltration into the CNS of T-cells and monocytes. EAE can beinduced in susceptible mice by immunization with proteolipid lipoprotein(PLP) in suitable adjuvants. The EAE animal model is an in vivo model ofhuman MS used to study pathogenic mechanisms of MS and to characterizenew agents for treating MS.

The F1Cs can be used in treating lung and airway conditions includingasthma conditions such as steroid independent asthma, severe asthma,atopic asthma, acute asthma or chronic asthma, allergic rhinitis,chronic bronchitis, acute bronchitis, cystic fibrosis, emphysema, lungfibrosis, lung airway hyperresponsiveness, chronic obstructive pulmonarydisease, pulmonary edema, pulmonary hypertension and acute respiratorydistress syndrome.

Exemplary compounds in the treatments include17α-ethynylandrost-5-ene-3β,7β,11β,17β-tetrol,17α-ethynylandrost-5-ene-3α,7β,11β,17β-tetrol,17α-ethynylandrost-5-ene-3β,7β,17β-triol,17α-ethynylandrost-5-ene-3β,7β, 11β,17β-tetrol,17β-ethynylandrost-5-ene-3β,7β,17α-triol,17α-ethynylandrost-5-ene-3α,7β,17β-triol,17α-ethynylandrost-5-ene-3β,7α,17β-triol,17α-ethynylandrost-5-ene-3β,17β-diol-7-one and analogs of thesecompounds where acetate ester or propionate ester is present at the3-position. Other analogs of these compounds include ones where R⁷ is—C₂H₅, e.g., 18-nor-18-ethyl-17α-ethynylandrost-5-ene-3β,7β, 11β,17β-tetrol, 18-nor-18-ethyl-17α-ethynylandrost-5-ene-3α,7β, 11β,17β-tetrol, 18-nor-18-ethyl-17α-ethynylandrost-5-ene-3β,7β,17β-triol,18-nor-18-ethyl-17α-ethynylandrost-5-ene-3α,7β,17β-triol,18-nor-18-ethyl-17α-ethynylandrost-5-ene-3β,7α,17β-triol and analogs ofany of these compounds wherein an ester such as acetate ester orpropionate ester is present at the 3-position. Other analogs of any ofthese compounds include ones wherein an ester such as acetate ester orpropionate ester is present at the 7-position or at both the 3- and7-positions, e.g., 3β-acetoxy-17α-ethynylandrost-5-ene-7β,17β-diol,3β-acetoxy-17α-ethynylandrost-5-ene-7β, 11β,17β-triol or7β-acetoxy-17α-ethynylandrost-5-ene-3β, 11β,17β-triol. Other compoundsare described herein.

Treatment of metabolic disorders. In some claimed embodiments, theformula 1 compounds are used to treat, prevent or slow the progressionof metabolic disorders such as type 1 diabetes, type 2 diabetes,Syndrome X, hypercholesterolemia, hyperglycemia, insulin resistance(e.g., associated with obesity or pre-diabetes), glucose intolerance,hypertriglyceridemia, hyperlipoproteinemia, a lipodystrophy condition,Syndrome X, arteriosclerosis, atherosclerosis and obesity. Syndrome X(including metabolic syndrome) is defined as a collection of two or moreabnormalities including hyperinsulemia, obesity, elevated levels oftriglycerides, uric acid, fibrinogen, small dense LDL particles andplasminogen activator inhibitor 1(PAI-1), and decreased levels of HDL-c.Many patients who have insulin resistance but have not yet developedtype 2 diabetes are also at a risk of developing metabolic syndrome,also referred to as syndrome X, insulin resistance syndrome orplurimetabolic syndrome. Syndrome-X typically occurs where a patient hastwo or more of hyperlipidemia, hyperinsulinemia, obesity, insulinresistance, insulin resistance leading to type-2 diabetes and diabeticcomplications thereof, i.e., diseases in which insulin resistance is thepart of the pathophysiology.

Independent risk factors have been associated with cardiovasculardisease associated with metabolic disorders can be treated with theF1Cs. These risk factors include hypertension, increased fibrinogenlevels, high levels of triglycerides, elevated LDL cholesterol, elevatedtotal cholesterol and low levels of HDL cholesterol. The treatment canresult in stimulation of pancreatic β-cells to secrete more insulinand/or a slowed rate of loss of pancreatic β-cells that can occur overtime in patients that have diabetes or that are obese.

In these claimed embodiments, treatment of metabolic disorders with aformula 1 compound can be combined with other treatments. Diabetes canbe treated with a formula 1 compound and one or more of a variety oftherapeutic agents including insulin sensitizers, such as PPAR-γagonists such as glitazones; biguanides; protein tyrosine phosphatase-1Binhibitors; dipeptidyl peptidase IV inhibitors; insulin; insulinmimetics; sulfonylureas; meglitinides; α-glucoside hydrolase inhibitors;and α-amylase inhibitors. Metformin, phenformin, acarbose androsiglitazone are agents that have been used to treat some type ofdiabetes.

As noted above, claimed embodiments may recite compositions containing aF1C to treat, prevent or slow the progression of insulin resistance orits symptoms. Insulin resistance is the diminished ability of insulin toexert its biological action across a broad range of concentrationsproducing less than expected biologic effect. Insulin resistant personshave a diminished ability to properly metabolize glucose and respondpoorly, if at all, to insulin therapy. Symptoms of insulin resistanceinclude insufficient insulin activation of glucose uptake, oxidation andstorage in muscle and inadequate insulin repression of lipolysis inadipose tissue and of glucose production and secretion in cells. Insulinresistance can cause or contribute to polycystic ovarian syndrome,impaired glucose tolerance, gestational diabetes, hypertension, obesityand atherosclerosis. These F1C compositions can be used to reducetriglyceride levels in patients who are insulin resistant.

As described above, the invention embodiments include a method toidentify a compound (or “test compound”) with a potential to treat, slowthe progression of, slow the onset of or ameliorate a metabolic disorderor a symptom thereof in a human or another mammal. The compoundsidentified by certain of the methods can be described as nonactivatorsof PPARs in vitro and incomplete NF-κB inhibitors in vitro that have oneor more of the described activities, which are typically obtained fromin vivo observations, e.g., delayed onset of hyperglycemia or slowedprogression of an existing diabetes condition. Compounds with thesecharacteristics are a new class of compounds that can be evaluated asagents to treat these disorders.

In these embodiments, the method comprises selecting a test compoundthat (i) does not activate one, two or three of PPAR-α, PPAR-γ andPPAR-δ in human or mammalian cells in vitro by more than about 10%,about 20%, about 30% or about 40% when compared to suitable negativecontrol human or mammalian cells in vitro; (ii) inhibits or decreasesthe transcriptional activity or level of NF-κB by about 20-80% or about25-75% or about 30-70% or about 35-65% in human or mammalian cells invitro when compared to suitable negative control human or mammaliancells in vitro; (iii) when compared to a suitable negative control ornormal control, decreases hyperglycemia, slows the progression or delaysthe onset of hyperglycemia, increases insulin sensitivity, decreasesglucose intolerance, slows the progression or rate of loss of pancreaticβ-islet cell numbers or their capacity to secrete insulin, increasespancreatic β-islet cell numbers or their capacity to secrete insulin,slows the rate of weight increase in db/db mice or mice with dietinduced obesity, decreases elevated levels of triglycerides, decreaseselevated levels total blood or serum cholesterol, decreases normal orelevated levels of LDL, VLDL, apoB-100 or apoB-48 in blood or serum orincreases normal or low levels of HDL or apoA1 in blood or serum ordecreases an elevated level of fibrinogen in blood or serum; and (iv)optionally, does not activate one or more of a glucocorticoid receptor,an androgen receptor an estrogen receptor-α, an estrogen receptor-β, amineralcorticoid receptor, a progesterone receptor or a biologicallyactive variant or isoform of any of these biomolecules in human ormammalian cells in vitro by more than about 5%, about 10%, about 20% orabout 30% when compared to suitable negative control human or mammaliancells in vitro. This permits identification or at least partialcharacterization of compounds with a potential to treat or amelioratethe metabolic disorder in the mammal.

In some embodiments, the activity of the test compound can be comparedto a suitable reference compound such as a formula 1 compound. Theformula 1 compound can be used in the method as a positive control or apositive reference standard that conforms to the characteristics themethod provides. Such compounds include17α-ethynylandrost-5-ene-3β,7β,17β-triol,17α-ethynylandrost-5-ene-3α,7β,17β-triol,17α-ethynylandrost-5-ene-7β,17β-diol-3-one andandrost-5-ene-3β,7β,16α,17β-tetrol. Other formula 1 compounds can beused as negative controls or reference standards that may exhibit none,one or two of the three required characteristics. Such compounds include16α-bromoepiandrosterone, 16α-bromo-3β,17β-dihydroxyandrost-5-ene and16α-hydroxyepiandrosterone.

Invention embodiments include determination of the effect of a testcompound on one or more conditions or symptoms associated with ametabolic disorder or disease. Typically such determinations arecompared to a suitable negative control or normal control or to asuitable positive control and the determination is conducted in a humanor an animal in vivo, although the determination can sometimes beconducted in vitro in whole cells or cell lysates. Drug products,described below, can incorporate or include information from suchdeterminations.

Decreases in hyperglycemia can be observed as a decrease in the level ofblood or serum glucose to a normal fasting range, which for humans atleast 2 years of age is about 70 mg/dL to 105 mg/dL or 115 mg/dL, withhyperglycemia being present at fasting glucose levels of about 135 mg/dLor about 140 mg/dL to 200 mg/dL, 300 mg/dL or 350 mg/dL. Glucose levelsabove about 400 mg/dL are life threatening. Postprandial glucose inblood or serum typically is measured at 2 hours after ingestion ofcarbohydrates, at least 75 g for humans, followed by a blood draw tomeasure glucose. Human glucose levels of 140 mg/dL to 200 mg/dL inpostprandial blood or serum indicate a hyperglycemia condition and aglucose level above 200 mg/dL identifies human diabetes mellitus. Forhumans, typically in patients having a normal fasting glucose level of70-115 mg/dL, an oral glucose tolerance test (OGTT) using blood can beconducted. In the OGTT for humans, if the peak glucose level (typicallyat 30 min or 1 hour after feeding) and 2 hour post carbohydrate valuesare above 200 mg/dL on two or more occasions, indicates that the patienthas diabetes mellitus.

A surrogate for blood glucose in humans is measurement of glycosylatedhemoglobin or Hb A1c, which is used, e.g., to monitor a diabetestreatment. Measurement of Hb A1c allows assessment of blood glucose orsugar levels over 100 to 120 days before the test and it is insensitiveto short term variations such as a recent meal or fasting state. Hb A1clevels of 2.2-48% are normal in adults, while levels of 2.5-5.9%indicate good control of diabetes, levels of 6-8% indicate fair diabetescontrol and levels above 8% Hb A1c indicate poor control of a diabetescondition. Procedures to conduct and interpret these and relatedprotocols have been described, e.g., K. D. Pagana and T. J. Pagana,Mosby's Diagnostic and Laboratory Test Reference, 5th edition, 2001,Mosby Inc., pages 441-448, 451-458, 507-509. Treatments with a formula 1compound in some embodiments can be monitored by observing decreased HbA1c, which correlates with improved diabetes treatment or improvedglucose control.

Practice of the claimed methods or other methods described herein canresult in normalization, e.g., return to levels within normal limits orranges or near normal limits or ranges of glucose, glucose surrogate orother values such as levels of phase reactive proteins or lipidcomponents such as total cholesterol, e.g., reduced LDL-cholesterol orincreased HDL-cholesterol. Normalization of glucose or surrogate valuesis typically observed as an elevated glucose or surrogate level droppingto within about 1%, about 2%, about 3% or about 5% of a normal glucoselevel or within about 5% or about 8% of a normal glucose surrogatevalue. Glucose values for other species have been described and similarmeasurements or assays can be used in the invention methods for thosespecies. Normalization of other values is typically observed as a returnof an abnormally high or low level to within about 2% or about 4% toabout 6%, about 10% or about 12% of the upper or lower end of thevalue's normal range for the subject species.

The compounds identified by the invention methods can be used to slowthe progression or delay the onset of hyperglycemia or to increaseinsulin sensitivity in insulin resistance where these exist or arereasonably expected to develop. Other effects of the compounds include adecreased glucose intolerance, slowed progression or rate of loss ofpancreatic β-islet cell numbers or their capacity to secrete insulin orincreased pancreatic β-islet cell numbers or capacity to secreteinsulin.

In some embodiments, the methods can be conducted in obese subjects.Obesity or “overweight” for humans as used herein generally refers to(1) an adult human male having a body mass index of about 26 kg/m², 27kg/m², 28 kg/m², 29 kg/m², 30 kg/m², 31 kg/m², 32 kg/m² or greater andadult human females having a body mass index of at least about 26 kg/m²,27 kg/m², 28 kg/m², 29 kg/m², 30 kg/m², 31 kg/m², 32 kg/m² or greater or(2) an obese or overweight condition as assessed by a health careprovider such as a physician or nurse. The determination of obesity for,e.g., a human, can take body fat content and distribution into account,since some persons with a high body mass index may not technically beobese due to a high amount of muscle tissue instead of fat or adiposetissue or due to a significant mounts of body fat or adipose in bodyareas other than the abdomen, e.g., hips or pelvis. Obesity and bodymass index has been described, e.g., G. A. Colditz, Med. Sci. SportsExerc., 31 (11), Suppl., pp. S663-S667, 1999, F. J. Nieto-Garcia et al.,Epidemiology, 1(2):146-152, 1990, R. H. Eckel, Circulation,96:3248-3250, 1999.

In some embodiments, the compounds identified by the invention methodsdo not significantly activate one or more of a mineralcorticoidreceptor, a progesterone receptor, a glucocorticoid receptor, anandrogen receptor an estrogen receptor-α, estrogen receptor-β or abiologically active variant of any of these biomolecules in human ormammalian cells in vitro by more than about 10%, about 20% or about 30%when compared to suitable negative control human or mammalian cells,typically as determined in and in vitro assay. Methods to measure theseactivities have been described, e.g., U.S. Pat. No. 5,298,429. In oneexemplary method, an assay for evaluating whether a test compound is afunctional ligand for a hormone receptor protein, or a functionalengineered or modified form thereof comprising: (a) culturing cellswhich contain: non-endogenous DNA which expresses the hormone receptorprotein, or functional engineered or modified form thereof, and DNAwhich encodes an operative hormone response element linked to a reportergene, wherein the culturing is conducted in the presence of at least onetest compound whose ability to function as a ligand or modulator for thehormone receptor protein, or functional engineered or modified formthereof, is sought to be determined, and (b) assaying for evidence oftranscription of said reporter gene in said cells. This assay willtypically be conducted using mammalian cells, e.g., CV-1 or COS cells.The reporter gene can be contained in a reporter plasmid where thenon-endogenous DNA expresses the hormone receptor protein or functionalmodified form thereof is contained in an expression plasmid, whereinsaid reporter and expression plasmids also contain the origin ofreplication of SV-40. Also, the reporter gene can be contained in areporter plasmid, wherein the non-endogenous DNA, which expresses thehormone receptor protein or functional modified form thereof, iscontained in an expression plasmid, where the reporter and expressionplasmids also contain a selectable marker. Related assays can use stablytransfected cells with detectable reporter genes, e.g., as described forestrogen receptor-β (ERβ-UAS-bla GripTite™ cell-based Assay, CatalogNumber K1091, Invitrogen Corp.), estrogen receptor-α (ERα-UAS-blaGripTite™ 293 cell-based Assay Catalog Number K1090, Invitrogen Corp.),androgen receptor (AR-UAS-bla GripTite™ 293 MSR cell-based Assay,Catalog Number K1082, Invitrogen Corp.) or progesterone receptor(Progesterone Receptor-UAS-bla HEK293T Assay, Catalog Number K1103,Invitrogen Corp.).

Claimed invention embodiments may include a method to identify orcharacterize a biological activity of a compound with a potential totreat or ameliorate a metabolic disorder in a mammal, comprisingselecting a compound that (i) does not activate one, two or three ofPPAR-α, PPAR-γ and PPAR-δ in human or mammalian cells in vitro by morethan about 30% when compared to suitable negative control human ormammalian cells in vitro; (ii) inhibits or decreases the transcriptionalactivity or level of NF-κB by about 20-80% in human or mammalian cellsin vitro when compared to suitable negative control human or mammaliancells in vitro; (iii) when compared to a suitable negative control ornormal control, decreases hyperglycemia, slows the progression or delaysthe onset of hyperglycemia, increases insulin sensitivity, decreasesglucose intolerance, slows the progression or rate of loss of pancreaticβ-islet cell numbers or their capacity to secrete insulin, increasespancreatic β-islet cell numbers or their capacity to secrete insulin,slows the rate of weight increase in db/db mice or in subjects with dietinduced or diet related obesity, decreases elevated levels oftriglycerides, decreases elevated levels total blood or serumcholesterol, decreases normal or elevated levels of LDL, VLDL, apoB-100or apoB-48 in blood or serum or increases normal or low levels of HDL orapoA1 in blood or serum or decreases an elevated level of fibrinogen inblood or serum; (iv) optionally, does not activate one or more of aglucocorticoid receptor, a mineralcorticoid receptor, a progesteronereceptor, an androgen receptor an estrogen receptor-α, estrogenreceptor-β or a biologically active variant of any of these biomoleculesin human or mammalian cells in vitro by more than about 30% whencompared to suitable negative control human or mammalian cells in vitro;and (v) optionally inhibits the level or activity of aphosphoenolpyruvate carboxykinase (PEPCK) or a 11β-hydroxysteroiddehydrogenase (11β-HSD), optionally 11β-HSD type 1 or 11β-HSD type 2 orthe level of a mRNA that encodes PEPCK or a 11β-HSD, in hepatocytes orliver-derived cells in vitro or in liver cells or tissue obtained fromliver cells or tissue in vivo; The method allows identification orcharacterization of the compound as having a potential to treat orameliorate the metabolic disorder in human or another mammal. The PEPCKenzyme can be cytosolic or mitochondrial in origin.

In some embodiments, the formula 1 compounds that are used arecharacterized by having a lack of appreciable androgenicity. In theseembodiments, the formula 1 compounds are characterized by having about30% or less, about 20% or less, about 10% or less or about 5% or less ofthe androgenicity of an androgen such as testosterone, testosteroneproprionate, dihydrotestosterone or dihydrotestosterone proprionate asmeasured in a suitable assay using suitable positive and/or negativecontrols. Suitable assays for androgenicity of various compounds havebeen described, e.g., J. R. Brooks, et al., Prostate 1991, 18:215-227,M. Gerrity et al., Int. J. Androl. 1981 4:494-504, S. S. Rao et al.,Indian J. Exp. Biol. 1969 7:20-22, O. Sunami et al., J. Toxicol. Sci.2000 25:403-415, G. H. Deckers et al., J. Steroid Biochem. Mol. Biol.2000 74:83-92. The androgenicity of the formula 1 compounds areoptionally determined as described or essentially as described in one ormore of these assays or any suitable assay.

Thus, one such embodiment comprises a method to treat a conditiondescribed herein comprising administering to a subject in need thereofan effective amount of a formula 1 compound, or delivering to thesubject's tissues an effective amount of a formula 1 compound, whereinthe formula 1 compound has about 30% or less, about 20% or less, about10% or less or about 5% or less of the androgenicity of an androgen suchas testosterone, testosterone proprionate, dihydrotestosterone ordihydrotestosterone proprionate as measured in a suitable assay, e.g.,as described in the citations above. In conducting such methods, thesubjects or mammals, e.g., rodents, humans or primates, are optionallymonitored for e.g., amelioration, prevention or a reduced severity of adisease, condition or symptom. Such monitoring can optionally includemeasuring one or more of cytokines (e.g., TNFα, IL-13, IL-1β), WBCs,platelets, granulocytes, neutrophils, RBCs, NK cells, macrophages orother immune cell types, e.g., as described herein or in the citedreferences, in circulation at suitable times, e.g., at baseline beforetreatment is started and at various times after treatment with a formula1 compound such as at about 2-45 days after treatment with a formula 1compound has ended.

Bone loss and repair conditions. Claimed embodiments may recite the useof a F1C or compositions containing a F1C and one or more excipients totreat, prevent, delay the onset of or slow the progression of bone loss,bone fracture or osteopenia disorders, e.g., an osteoporosis conditionsuch as primary osteoporosis, postmenopausal or type 1 osteoporosis,involutional or type 2 osteoporosis, idiopathic osteoporosis, asecondary osteoporosis such as a glucocorticoid associated bone losscondition and bone loss associated with a trauma such as a first, secondor third degree thermal, chemical or radiation burn. These treatmentscan improve bone mass, bone density and/or bone strength over time.

Drug products. In some embodiments, the invention provides a drugproduct for treating an inflammation, autoimmune or other conditiondescribed herein. The drug product typically comprises (a) the drug in adosage form such as a solid or liquid formulation suitable for, e.g.,oral or parenteral administration. Packaging for the drug and/or apackage insert or label will have information about the drug's efficacy,mechanism of action, the intended patient population, dosage, doseregimen, route of administration, toxicity of the biological insult orthe severity of insult that the drug can be used to treat, if this isknown. When the biological insult is radiation exposure, the packageinsert or label can contain information about the radiation dose or doserange for which the drug product can be used or is approved. The drugproduct can optionally contain a diary or use instructions for thepatient to record when or how the drug is used or what symptoms or drugeffects the drug user experiences during or after use of the drug. Thiscan be used to aid in phase IV or post marketing analyses of the drug'sefficacy or side effects. Other embodiments of drug products are asdescribed in other embodiments described herein.

A drug product as used herein means a product that has been reviewed andapproved for marketing or sale by a regulatory agency or entity withauthority to review or approve applications for sale or medical use,e.g., the U.S. Food and Drug Administration or the European MedicinesAgency or European Medicines Evaluation Agency. Uses of drug productsinclude its marketing or sales and offers to sell or buy it forconsideration. These activities will typically adhere to terms of theregulatory approval that may affect or govern marketing, sales,purchases or product handling. The drug in a drug product can be a newdrug, a generic drug, a biological, a medical device or a protocol forthe use of any of these. The drug product usually results from marketingapproval by the U.S. Food and Drug Administration or by the EuropeanMedicines Evaluation Agency of a U.S. or non-U.S. new drug application,an abbreviated new drug application, a biological license application oran application to market a medical device. Uses for the drug productinclude its sale to public or private buyers such as the U.S. Departmentof Defense, the U.S. Department of Energy, U.S. Department of Health andHuman Services or a private drug buyer or distributor entity. Other usesinclude use of the drug to treat indicated or approved medicalconditions and physician approved uses or off label uses. Pre-approvaldrug products are other aspects of the invention, which may beessentially the same as drug products described herein, but it can beused to prepare a drug or regulatory submission for marketing or forregulatory review before marketing approval.

The intended patient population identified by the drug product can alsospecify excluded populations, if any that may apply such as pediatricpatients or elderly patients. Information about dosage will typicallyspecify daily doses of the drug, while the dose regimen will describehow often and how long the drug is to be administered or taken. Theroute of administration will identify one or more routes that aresuitable for use of the drug, although a given formulation willtypically be approved for only one route of administration. Dosages,dose regimens and routes of administration that the package or label mayidentify are described elsewhere herein.

In one embodiment, the drug product is for treatment, prevention oramelioration of an inflammation condition or another condition describedherein and it comprises or includes a formulation that contains acompound such as a formula 1 compound formulated with 1, 2, 3, 4 or moreexcipient(s) for oral or parenteral administration, e.g., intramuscular,subcutaneous or subdermal injection, with a package insert or labeldescribing administration of a daily dose of, e.g., about 0.01 mg, 0.05mg, 0.1 mg, 0.5 mg, 1 mg, 4 mg, 5 mg, 10 mg, 20 mg, 25 mg, 40 mg, 50 mg,80 mg, 100 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 300 mg, 350 mg,400 mg, 450 mg or 500 mg of a formula 1 compound for 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more consecutive days beginning after the disease orcondition is diagnosed or otherwise observed. Information that thepackage insert or label can contain includes information aboutbiological responses to the drug or the treatment regimen. Theinformation can include a description of one or more of (a) one or moreside-effects or toxicities associated with use of the drug in humans ormammals such as non-human primates, (b) its effect on the inflammationor other condition, e.g., in a protocol or suitable variation describedherein, (c) protocols or instructions for the use of additionaltherapeutic agents such as dexamethasone or other glucocorticoids withthe drug and (d) the time or time period when administration of the drugshould begin for best or known therapeutic benefit.

T cell subset regulation. In some aspects, the invention provides amethod to identify a compound with a potential to detectably modulatethe numbers or activity of CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁺CD103⁺regulatory T cells, CD4⁺CD25^(high)CD103⁺ regulatory T cells orCD4⁺CD25^(high) regulatory T cells in a mammal, comprising selecting acompound that (i) does not activate or inhibit one or more of aglucocorticoid receptor, an androgen receptor an estrogen receptor-α,estrogen receptor-β or a biologically active variant of any of thesebiomolecules in human or mammalian cells in vitro by more than about 20%or about 30% when compared to suitable control human or mammalian cellsin vitro; (ii) has a molecular weight of about 100-1000 Daltons,optionally a molecular weight of about 250-850 Daltons; (iii) whencompared to a suitable negative control or normal control, increases ordecreases the numbers or activity of CD4⁺CD25⁺ regulatory T cells,CD4⁺CD25+CD103⁺ regulatory T cells, CD4⁺CD25^(high)CD103⁺ regulatory Tcells or CD4⁺CD25^(high) regulatory T cells by more than 20% in asuitable assay; and (iv) optionally inhibits or decreases thetranscriptional activity or level of NF-κB by about 20-80% in human ormammalian cells in vitro when compared to suitable negative controlhuman or mammalian cells in vitro. The formula 1 compounds and othercompounds can be used in these embodiments essentially as described inexamples 20 or 21 below.

Compounds in vitro or in vivo that increase or decrease the activity ornumbers of certain T cell subsets such as CD4⁺CD25⁺ T cells andCD4⁺CD25^(high)T cells are candidates for treating or slowing the onsetor progression of autoimmune conditions, cancer, neurological trauma ordisorders such as neuron loss after a trauma such as ischemia andmetabolic diseases such as type I diabetes, atherosclerosis, cell, organor tissue rejection in autologous transplantations and graft versus hostdisease in situations there these conditions exist or may occur. Thetreatments can be used for improving wound healing, treating reperfusioninjury, stenosis, restenosis after angioplasty, myocardial or cerebralinfarction. Embodiments include compounds having a molecular weight ofless than about 2,000 Daltons, less than about 1,000 Daltons or lessthan about 500 Daltons. One group of compounds has a molecular weight ofabout 285 or 290 to about 500 or 650 Daltons. Treg cell responses can beobserved as an increase or decrease of about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 60%, about 70%, about 80%, about 100%, about 200%, about400%, about 600%, about 1000%, about 2000%, about 5000%, about 10000% ormore in the numbers of Treg cells, typically CD4⁺CD25⁺ T cells orCD4⁺CD25^(high) T cells, in subjects or in in vitro assays treated withcompound compared to suitable negative controls. These changes can beobserves as increases or decreases in Treg cell numbers or theiractivity in circulating blood or in cells in vitro or in vivo.

Methods to analyze subset cell profiles such as T cell profiles can beobtained by any of a variety of methods including flow cytometry (FACS),for example, Levy et al., Clin. Immunol. Immunopathol. 35:328, 1985. InFACS analysis, monoclonal antibodies to a variety of subset cells bindto and identify phenotypic surface antigens that are present on thecells. Commercially available antibodies exist that can detect thepresence of these markers, so that preparation of the antibodies isgenerally not required. Antibodies that identify the same or a closelylinked antigenic marker would be expected to give similar diagnosticresults. Thus, where a marker antigen is designated in the specificationor claims by reference to a particular monoclonal antibody with which itbinds, e.g., CD4 or CD25, such a designation includes that marker evenif different monoclonal antibodies are used in the identification.Phenotypic markers of interest include general markers for varioussubset cell types including CD3 for total T cells, CD4 for Thelper/inducer cells, CD8 for T suppressor/cytotoxic cells, and CD16/56for NK cells; CD8-expressing subset markers such as CD11b for Tsuppressor cells, CD38 for activated T suppressor/cytotoxic cells,HLA-DR for activated T suppressor/cytotoxic cells, and CD57; and CD4expressing markers such as CD25 and HLA-DR for activated Thelper/inducer cells, including Treg cells.

Dosing protocols or methods. In treating any of the conditions orsymptoms disclosed herein, one can continuously (daily) orintermittently administer the formula 1 compound(s) to a subjectsuffering from or susceptible to the condition or symptom. In treating acondition such as an inflammation condition or another conditiondisclosed herein with a formula 1 compound intermittent dosing couldavoid or ameliorate some of the undesired aspects normally associatedwith discontinuous dosing. Such undesired aspects include failure of thepatient or subject to adhere to a daily dosing regimen or reduction ofthe dosages of other therapeutic agents such as glucocorticoids and/ortheir associated unwanted side effects or toxicities such as bone lossor resorption.

In some embodiments, daily dosing will continue as long as the diseaseor symptoms are apparent, typically for chronic conditions. In otherembodiments, daily dosing will continue for 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 consecutive days and then be followed by a period of no dosing untilor if dosing is again needed. These embodiments will typically involvetreating acute conditions that may or may not recur from time to time.Treatment of chronic conditions will typically involve continuous dailydosing for extended periods of time.

In any of continuous (daily) or intermittent dosing regimen, or intreating any of the diseases, conditions or symptoms described herein,the formula 1 compound(s) can be administered by one or more suitableroutes, e.g., oral, buccal, sublingual, topical, intramuscular,subcutaneous, subdermal, intravenous, intradermal or by an aerosol.

The daily dose is usually about 0.001 mg/kg/day to about 200 mg/kg/day.Typical dose ranges are about 0.1 to about 100 mg/kg/day, includingabout 0.2 mg/kg/day, 0.5 mg/kg/day, about 1 mg/kg/day, about 2mg/kg/day, about 4 mg/kg/day, about 5 mg/kg/day or about 6 mg/kg/day.One can administer the formula 1 compound(s) orally or by parenteraladministration using about 2 to about 50 mg/kg/day or about 2-40mg/kg/day. Such dosing will typically give a serum level of the formula1 compound of about 1 mg/mL, about 4 mg/mL or about 8 mg/mL to about 125mg/mL or about 250 mg/mL, e.g., about 15 mg/mL to about 120 mg/mL orabout 20 mg/mL to about 100 mg/mL. Such a serum level can be transient,e.g., lasting about 30 minutes or about 60 minutes to about 2 hours orabout 8 hours, which will may occur on days when the compound isadministered or at later time for depot formulations. For humans orother mammals an oral or parenteral daily dose will typically be about0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 4 mg, 5 mg, 10 mg, 20 mg, 25 mg,40 mg, 50 mg, 80 mg, 100 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 300mg, 350 mg, 400 mg, 450 mg or 500 mg of a formula 1 compound, which canbe present as a unit dosage, e.g., tablets, capsules, or other forms fororal administration. Such daily doses can often be about 5 mg/day toabout 250 mg/day.

Continuous daily dosing is usually used to treat the chronic conditionsdescribed herein. Daily doses are usually given as a single dose, butdaily doses can be subdivided into 2 or 3 subdoses. Intermittent dosingprotocols include administration of a formula 1 compound every other dayor every third day for a suitable time period. When treating blood celldeficiencies dosing will usually begin on the same day that the subjectexperiences a short-lived myeloablative event such as a radiationexposure. For longer lasting events, e.g., cancer chemotherapy, dosingwith the formula 1 compound can begin at about 12 hours, about 1 day,about 2 days or 3 about days after a chemotherapy agent has beenadministered to the subject. Daily dosing can continue for definedperiods followed by no dosing for a fixed or variable period of time. Inthese embodiments, a disease flare such as a multiple sclerosis, opticneuritis, arthritis, asthma, a colitis condition such as ulcerativecolitis or Crohn's disease flare can be treated by daily dosing forabout 3, 5, 7, 14 or 28 consecutive days, followed by no furthertreatment until another flare occurs or begins.

Clinical conditions and symptoms. Claimed embodiments may recite thecompounds and methods described herein to treat, ameliorate, prevent orslow the progression of conditions described herein and/or one or moreof their symptoms. Such uses include inhibiting bone resorption,decreasing unwanted side effects associate with or caused by achemotherapy, e.g., antiinflammatory glucocorticoids. Unwantedinflammation conditions include lung inflammation conditions, e.g., lungfibrosis, emphysema, cystic fibrosis, acute or chronic asthma, bronchialasthma, atopic asthma, ARDS or COPD, or autoimmune disorders such asosteoarthritis, rheumatoid arthritis, a pancreatitis such as autoimmunepancreatitis, systemic lupus erythematosis, lupus erythematosus-relatedtissue inflammation, lupus erythematosus-related arthritis, lupuserythematosus-related skin changes, lupus erythematosus-relatedhematologic abnormalities, lupus erythematosus-related kidneyimpairment, lupus erythematosus-related heart or lung disease, andunwanted lupus erythematosus-related neuropsychiatric or neurologicalchanges.

Symptoms of conditions that can be treated include fever, joint pain(arthralgias), arthritis, and serositis (pleurisy or pericarditis).Administration of other agents can also be used in the presenttreatments. Thus, pain can be treated using nonsteroidal,anti-inflammatory drugs, such as aspirin, salisylates, ibuprofen,naproxen, clinoril, oxaprozin and tolmetin. Cutaneous features ofsystemic lupus can be treated with antimalarial drugs, such ashydroxychloroquine, chloroquine and quinacrine. Retinoids such asistretinoin and etretinate can also be used to treat skin symptoms incombination with the compounds described herein. Organ damage can betreated with corticosteroids, usually given orally or intravenously.Corticosteroids that can be used include hydrocortisone (cortisol),corticosterone, aldosterone, ACTH, triamcinolone and derivatives such astriamcinolone diacetate, triamcinolone hexacetonide, and triamcinoloneacetonide, betamethasone and derivatives such as betamethasonedipropionate, betamethasone benzoate, betamethasone sodium phosphate,betamethasone acetate, and betamethasone valerate, flunisolide,prednisone and its derivatives, fluocinolone and derivatives such asfluocinolone acetonide, diflorasone and derivatives such as diflorasonediacetate, halcinonide, dexamethasone and derivatives such asdexamethasone dipropionate and dexamethasone valerate, desoximetasone(desoxymethasone), diflucortolone and derivatives such as diflucortolonevalerate), fluclorolone acetonide, fluocinonide, fluocortolone,fluprednidene, flurandrenolide, clobetasol, clobetasone and derivativessuch as clobetasone butyrate, alclometasone, flumethasone, andfluocortolone.

When oral administration of corticosteroids is insufficient, intravenousmethyl prednisolone pulse therapy (high dose) can be used to treat lupusnephritis and other serious non-renal manifestations, such as hemolyticanemia, central nervous system inflammation (cerebritis), low-plateletcounts, and severe pleuropericarditis.

The formula 1 compounds can be used to treat, prevent or slow theprogression of osteoporosis or bone fractures. The treatment of subjectscan lead to strengthening of bones and/or reduced loss of bone mass orminerals, resulting in increased resistance to fractures. As usedherein, “treating” conditions such as those described herein means thatthe treatment can result in amelioration, prevention or slowedprogression of the conditions, and/or amelioration, prevention or slowedprogression of one or more symptoms of such conditions.

Formulations and compositions for preparing formulations. Claimedinvention embodiments may include formulations described here andelsewhere in this disclosure. While it is possible for the formula 1compound(s) to be administered alone it is usual to present them asformulations. The formulations, both for veterinary and for human use,comprise at least one formula 1 compound, together with one or moreexcipients and optionally one or more additional therapeuticingredients. Usually only one F1C is present in the composition, withonly low, trace or essentially undetectable levels (less than about 3%by weight or less than about 2% of total F1C) of other F1Cs, e.g., oneor more epimers of the primary F1C.

Formulations include compositions comprising 1, 2, 3, 4 or morepharmaceutically acceptable excipients or carriers. The compositions areused to prepare formulations suitable for human or animal use. Suitableadministration routes for formulations include oral, rectal, nasal,topical (including buccal and sublingual), vaginal, rectal andparenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal, intraocular and epidural). In general, aqueousand non-aqueous liquid or cream formulations are delivered by aparenteral, oral or topical route. In other embodiments, such as theinvention intermittent dosing methods, the formula 1 compound(s) may bepresent as an aqueous or a non-aqueous liquid formulation or a solidformulation suitable for administration by any of the routes disclosedherein, e.g., oral, topical, buccal, sublingual, parenteral, inhaledaerosol or a depot such as a subcutaneous depot or an intraperitoneal orintramuscular depot. It will be appreciated that the preferred route mayvary with, for example, the subject's pathological condition or weightor the subject's response to therapy with a formula 1 compound or othertherapy that is used or that is appropriate to the circumstances.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods known in the art ofpharmacy. Techniques, excipients and formulations generally are foundin, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa. 1985, 17^(th) edition, Nema et al., PDA J. Pharm. Sci. Tech.1997 51:166-171, G. Cole, et al., editors, Pharmaceutical CoatingTechnology, 1995, Taylor & Francis, ISBN 0 136628915, H. A. Lieberman,et al., editors, Pharmaceutical Dosage Forms, 1992 2^(nd) revisededition, volumes 1 and 2, Marcel Dekker, ISBN 0824793870, J. T.Carstensen. Pharmaceutical Preformulation, 1998, pages 1-306, TechnomicPublishing Co. ISBN 1566766907. Exemplary excipients for formulationsinclude emulsifying wax, propyl gallate, citric acid, lactic acid,polysorbate 80, sodium chloride, isopropyl palmitate, glycerin, whitepetrolatum and other excipients disclosed herein.

Formulations, or compositions disclosed herein for use to makeformulations suitable for administration by the routes disclosed hereinoptionally comprise an average particle size in the range of about 0.01to about 500 microns, about 0.1 to about 100 microns or about 0.5 toabout 75 microns. Average particle sizes include a range between 0.01and 500 microns in 0.05 micron or in 0.1 micron or other increments,e.g., an average particle size of about 0.05, 0.1, 0.5, 1, 1.5, 2.0,2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 50, 60, 75, 85, 100, 120, etc. microns). When formula 1 compounds orcompositions that comprise a formula 1 compound are used asintermediates to make a formulation, they may comprise one, two, threeor more of these average particle sizes, or size ranges. In preparingany of the compositions or formulations that are disclosed herein andthat comprise a formula 1 compound (and optionally one or moreexcipients), one may optionally mill, sieve or otherwise granulate thecompound or composition to obtain a desired particle size.

Thus, one such embodiment comprises a method to treat a conditiondescribed herein comprising administering to a subject in need thereofan effective amount of a formula 1 compound, or delivering to thesubject's tissues an effective amount of a formula 1 compound, whereinthe formula 1 compound has about 30% or less, about 20% or less, about10% or less or about 5% or less of the androgenicity of an androgen suchas testosterone, testosterone proprionate, dihydrotestosterone ordihydrotestosterone proprionate as measured in a suitable assay, e.g.,as described in the citations above. In conducting such methods, thesubjects or mammals, e.g., rodents, humans or primates, are optionallymonitored for e.g., amelioration, prevention or a reduced severity of adisease, condition or symptom. Such monitoring can optionally includemeasuring one or more of cytokines (e.g., TNFα, IL-13, IL-1β), WBCs,platelets, granulocytes, neutrophils, RBCs, NK cells, macrophages orother immune cell types, e.g., as described herein or in the citedreferences, in circulation at suitable times, e.g., at baseline beforetreatment is started and at various times after treatment with a formula1 compound such as at about 2-45 days after treatment with a formula 1compound has ended.

As noted above, in some embodiments a treatment with a formula 1compound is combined with a corticosteroid or glucocorticoid.Corticosteroids are used in a number of clinical situations to, e.g.,decrease the intensity or frequency of flares or episodes ofinflammation or autoimmune reactions in conditions such as acute orchronic rheumatoid arthritis, acute or chronic osteoarthritis, a colitiscondition such as ulcerative colitis, acute or chronic asthma, bronchialasthma, psoriasis, systemic lupus erythematosus, hepatitis, pulmonaryfibrosis, type I diabetes, type II diabetes or cachexia. However, manycorticosteroids have significant side effects or toxicities that canlimit their use or efficacy. The formula 1 compounds are useful tocounteract such side effects or toxicities without negating all of thedesired therapeutic capacity of the corticosteroid. This allows thecontinued use, or a modified dosage of the corticosteroid, e.g., anincreased dosage, without an intensification of the side effects ortoxicities or a decreased corticosteroid dosage. The side-effects ortoxicities that can be treated, prevented, ameliorated or reducedinclude one or more of bone loss, reduced bone growth, enhanced boneresorption, osteoporosis, immunosuppression, increased susceptibility toinfection, mood or personality changes, depression, headache, vertigo,high blood pressure or hypertension, muscle weakness, fatigue, nausea,malaise, peptic ulcers, pancreatitis, thin or fragile skin, growthsuppression in children or preadult subjects, thromboembolism,cataracts, and edema. Dosages, routes of administration and dosingprotocols for the formula 1 compound would be essentially as describedherein. An exemplary dose of formula 1 compound of about 0.5 to about 20mg/kg/day is administered during the period during which acorticosteroid is administered and optionally over a period of about 1week to about 6 months or more after dosing with the corticosteroid hasended. The corticosteroids are administered essentially using knowndosages, routes of administration and dosing protocols, see, e.g.,Physicians Desk Reference 54^(th) edition, 2000, pages 323-2781, ISBN1-56363-330-2, Medical Economics Co., Inc., Montvale, N.J. However, thedosage of the corticosteroid may optionally be adjusted, e.g., increasedabout 10% to about 300% above the normal dosage, without a correspondingincrease in all of the side effects or toxicities associated with thecorticosteroid. Such increases would be made incrementally over asufficient time period and as appropriate for the subject's clinicalcondition, e.g., daily corticosteroid dose increases of about 10% toabout 20% to a maximum of about 300% over about 2 weeks to about 1 year.

The treatment method can be used to, treat, prevent or ameliorate anacute trauma such as a myocardial infarction, a hemorrhage such as acerebral hemorrhage or stroke or a bone fracture, osteoporosis or excessor unwanted bone resorption or loss. The treatments can be used tofacilitate repair of damage or injury to skin, mucosa, cartilage, liver,heart tissue, bone or CNS or neural tissue in situations where there isdamage, e.g., chemical or heat burns, osteoarthritis, rheumatoidarthritis, liver cirrhosis, osteoporosis, bone fracture, myocardialinfarction, stroke or head trauma. The treatments can also be used toreduce bone loss due to a therapy, e.g., a glucocorticoid therapy in alupus condition or in patients having an inflammatory bowel disease,Crohn's disease, acute or chronic colitis or a renal disorder such asacute or chronic renal failure or autoimmune renal injury.

The following embodiments describe one or more aspects of the invention.

1. A method to identify or characterize a biodynamic compoundcomprising, measuring a biological response of a test compound in vivoin a subject after exposure of the subject to an acute stimulus orbiological insult that elicits a detectable response to the acutestimulus or biological insult, wherein the test compound elicits afavorable treatment response on a mediator of the acute biologicalresponse to the stimulus or biological insult at a time or time periodwhen (i) the acute response is maximal or nearly maximal or (ii) theacute response is increasing in a period of a prolonged acute biologicalresponse and wherein the favorable treatment response differs at time(i) or (ii) from its effect on the mediator of the acute biologicalresponse at one, two, three or more earlier or later times or timeperiods and such effect at the earlier or later times or time periods isan increase or decrease of less than about 50% in the level or activityof the mediator of the acute biological response when compared tosuitable vehicle or placebo controls at the same or essentially the sameearlier or later times or time period, whereby a compound that elicits afavorable treatment response on the mediator of the acute biologicalresponse and the favorable treatment response differs at time (i) or(ii) from its effect on the mediator of the acute biological response atone, two, three or more earlier or later times or time periods isidentified as a biodynamic compound.

2. The method of embodiment 1 further comprising conducting a protocolto determine if the test compound modulates the activity or level of themediator of the acute biological response by about 25% to about 75% inan assay in vitro, optionally wherein the test compound does notactivate or antagonize a glucocorticoid receptor by more than about 20%when compared to a suitable reference activator or antagonist of theglucocorticoid receptor.

3. The method of embodiment 1 or 2 wherein the acute stimulus orbiological insult is exposure of the subject to a sufficient amount ofionizing radiation or a proinflammatory signal, compound or composition,optionally wherein the proinflammatory signal, compound or compositionis bacterial LPS or TNFα, and/or optionally wherein the mediator of theacute biological response is NF-κB or IκB.

4. The method of embodiment 1, 2 or 3 wherein the acute stimulus orbiological insult is administration of sufficient bacterial LPS to asufficient number of drug treated mice and a sufficient number vehiclecontrol mice and measurement of the effect of the test compound on themediator of the acute biological response at a time when (i) the acuteresponse is maximal or nearly maximal, optionally at about 1.5 hoursafter administration of bacterial LPS by intraperitoneal injection and(ii) one or two other time points before and/or after the administrationof the sufficient bacterial LPS, optionally at one time point before theadministration of the sufficient bacterial LPS and at one later timeafter the acute response is maximal or nearly maximal, optionally atabout 2.0 or 2.5 hours after administration of bacterial LPS byintraperitoneal injection, and optionally wherein the mediator of theacute biological response is NF-κB or IκB.

5. The method of embodiment 4 wherein the administration of sufficientbacterial LPS is accomplished essentially according to the methoddescribed at example 9 or a suitable variation thereof and optionallywherein the capacity of the compound to partially modulate the level oractivity of the mediator of the acute biological response isaccomplished essentially according to the method described at example 7or a suitable variation thereof.

6. The method of embodiment 1, 2, 3, 4 or 5 comprising inclusion of apositive biodynamic drug control, optionally17α-ethynylandrost-5-ene-3β,7β,17β-triol, to assess the relative potencyor efficacy of the test compound and optionally including biostatic drugcontrol to assess the relative potency or efficacy of the test compound.

7. A drug product or pre-approval drug product comprising a drug in adosage form and packaging for the drug together with a package insert orlabel that includes information about the drug's efficacy, mechanism ofaction or clinical use, wherein the efficacy, mechanism of action orclinical use information was obtained at least in part from acharacterization method that comprises the method of embodiment 1, 2, 3,4 or 5.

8. A drug product or pre-approval drug product comprising a drug in adosage form and packaging for the drug together with a package insert orlabel that includes information about the drug's efficacy, mechanism ofaction or clinical use, wherein the efficacy, mechanism of action orclinical use information was obtained at least in part from acharacterization method that comprises (a) contacting a cell or cells invitro for a sufficient time with a sufficient amount of an activator ofNF-κB activity wherein the cell(s) can respond to the activator of NF-κBby detectably increasing the level or activity of NF-kB in the cell(s);(b) contacting the cell(s) in vitro for a sufficient time with asufficient amount of the drug, wherein the drug detectably inhibits theactivation of NF-κB activity compared to suitable control; and (c)optionally comparing the drug's capacity to inhibit activation of NF-kBwith a reference compound, wherein the reference compound is a formula 1compound described herein that has the capacity to detectably inhibitactivation of NF-κB in the characterization method by about 25% to about75%, wherein the drug inhibits activation of NF-kB by about 25% to about75% in the characterization method and optionally wherein the referencecompound or the drug does not detectably or significantly bind directlyto a glucocorticoid receptor or optionally wherein the referencecompound or the drug does not detectably or significantly agonize aglucocorticoid receptor, optionally the drug does not agonize aglucocorticoid receptor by more than about 20% compared to a suitableagonist control.

9. The drug product of embodiment 7 or 8 wherein the dosage formcomprises an oral, parenteral, topical or inhalation formulation.

10. The drug product of embodiment 8 or 9 wherein the reference compoundor the drug inhibits activation of NF-kB by about 35% to about 70% or byabout 40% to about 65% in the characterization method.

11. The drug product of embodiment 8, 9 or 10 wherein the NF-κB in thecells is activated by one, two, three or more of TNF-α, TNF-β, TGF-β,IL-1, epidermal growth factor, bacterial LPS, bacterial peptidoglycan,yeast zymosan, bacterial lipoprotein, a bacterial or viral antigen orgene product, ultraviolet irradiation, heat or a temperature increase, alymphokine or an oxidant free radical, or H₂O₂.

12. The drug product of embodiment 8, 9, 10 or 11 wherein the referencecompound or the drug binds directly to a glucocorticoid receptor with ak_(d) of >10 μM in a suitable binding assay or wherein the referencecompound or the drug does not detectably agonize a glucocorticoidreceptor at a concentration of equal to or greater than about 10 μM inan assay suitable to detect activation or an increase of glucocorticoidreceptor-mediated gene expression.

13. The drug product of embodiment 8, 9, 10, 11 or 12 wherein thecell(s) in vitro are mammalian, rodent or human cell(s) optionallyselected from the group consisting of human THP-1 cells, rat RAW cells,macrophages, monocytes, T-lymphocytes, B-lymphocytes, dendritic cells,glial cells, Kupfer cells, hepatocytes, neutrophils, white blood cellsand cells from whole blood.

14. The drug product of embodiment 7 or 8 wherein the information aboutthe drug's efficacy, mechanism of action or clinical use is included ina submission to a regulatory agency or a review entity with authority toreview or approve the commercial use or marketing of the drug product.

15. A method to treat an inflammation condition or autoimmune disease ina mammal, comprising administering to the subject, or delivering to thesubject's tissues, an effective amount of a biodynamic compoundidentified by the method of embodiment 1, 2, 3, 4, 5 or 6, wherein apositive biodynamic compound is used as a reference standard or whereinthe biodynamic compound partially inhibits the mediator of the acutebiological response in a suitable assay in vitro, wherein the suitableassay in vitro optionally is essentially according to the method ofexample 7 or a suitable variation thereof.

16. A compound having the structure

wherein one R¹ is —H or C₁₋₈ optionally substituted alkyl and the otherR¹ is —OH, a C₂₋₈ ester or a C₁₋₈ ether or both R¹ together are ═O; oneR² is —H or C₁₋₈ optionally substituted alkyl and the other R² is —H,—OH, a C₂₋₈ ester or a C₁₋₈ ether or both R² together are ═O; one R³ is—H or C₁₋₈ optionally substituted alkyl and the other R³ is —OH, a C₂₋₈ester, a C₁₋₈ ether or C₁₋₈ optionally substituted alkyl; one R⁴ is —Hor C₁₋₈ optionally substituted alkyl, preferably —CH₃, —C≡CH or —C≡C—Cl,and the other R⁴ is —OH, a C₂₋₈ ester or a C₁₋₈ ether; R⁵ is —CH₃, —C₂H₅or —CH₂OH; R⁵ is —H, —CH₃, —C₂H₅ or —CH₂OH; one R⁷ is —H or C₁₋₈optionally substituted alkyl and the other R⁷ is —H, —OH, a C₂₋₈ esteror a C₁₋₈ ether; R¹⁰ is —H or a halogen; and one R¹¹ is —H or C₁₋₈optionally substituted alkyl and the other R¹¹ is —H, —OH, a C₂₋₈ ester,a C₁₋₈ ether or C₁₋₈ optionally substituted alkyl. The compound(s) inthis embodiment and those below can be partially purified, e.g., ≧ about70% or ≧ about 80% pure by weight compared to other F1Cs, or more highlypurified, ≧ about 90% or ≧ about 95% or ≧ about 97% pure by weightcompared to other F1Cs. These compounds can be used in one or more ofthe embodiments described herein.

17. The compound according to embodiment 16 wherein one, two or three ofR², R⁷ or R¹¹ is —OH, a C₂₋₈ ester, a C₁₋₈ ether, ═O or ═NOH.

18. The compound according to embodiment 16 or 17 selected from17α-ethynylandrost-5-ene-3β,7β,17β-triol,17α-ethynylandrost-5-ene-3α,7β,17β-triol,17α-ethynylandrost-5-ene-3β,17β-diol-7-one,17α-ethynylandrost-5-ene-7β,17β-diol-3-one,17α-ethynylandrost-5-ene-3β,7β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3α,7β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3β,7α,16α,17β-tetrol,17α-ethynylandrost-5-ene-3β,4β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3α,4β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3β,11β,16α,17β-tetrol,17β-ethynylandrost-5-ene-3α,11β,16α,17α-tetrol,17β-ethynylandrost-5-ene-3α,11β,16β,17α-tetrol,androst-5-ene-3α,11β,16β,17β-tetrol or a C₂₋₄ monoester or C₂₋₄ diesteranalog of any of these compounds, optionally wherein (1) the C₂₋₄monoester is acetate or propionate at the 3- or 17-position or (2) theC₂₋₄ diester is acetate or propionate at the 3- and 17-positions. Otheranalogs include compounds wherein the ethynyl moiety at the 17-positionis replaced with chloroethynyl, e.g.,17α-chloroethynylandrost-5-ene-3β,7β,17β-triol,17α-chloroethynylandrost-5-ene-3α,7β,17β-triol and17α-chloroethynylandrost-5-ene-7β,17β-diol-3-one.

19. The compound according to embodiment 17 or 18 wherein the compoundis (a) a powder or granule that is at least 80% pure, at least 95% pureor at least 98% pure or (b) a solution or suspension that is at least80% pure, at least 95% pure or at least 98% pure. These compoundsinclude 17α-ethynylandrost-5-ene-3β,7β,17β-triol,androst-5-ene-3β,4β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3β,4β,16α,17β-tetrol,androst-5-ene-3β,11β,16α,17β-tetrol, androst-5-ene-3β,7β,16α,17β-tetroland epimers of these compounds wherein the configuration of one or twohydroxyl groups is changed from α- to β- or from β- to α-, e.g.,17β-ethynylandrost-5-ene-3β,7β,17α-triol,17β-ethynylandrost-5-ene-3β,4β,16α,17α-tetrol,17α-ethynylandrost-5-ene-3α,7β,17β-triol or17α-ethynylandrost-5-ene-3α,4β,16α,17β-tetrol.

20. The compound according to embodiment 17, 18 or 19 wherein thecompound is about 80%, about 85%, about 90%, about 95%, about 97% orabout 98% to about 99.5% or about 99.9% pure, optionally wherein thecompound is in the form of a powder or granules, optionally wherein thepowder has an average particle size of about 50 nm or about 100 nm toabout 5 μm, about 10 μm or about 25 μm as measured in a suitable assaysuch as light scattering.

21. A method to identify a compound with a potential to detectablymodulate the numbers or activity of CD4⁺CD25⁺ regulatory T cells,CD4⁺CD25⁺CD103⁺ regulatory T cells, CD4⁺CD25^(high)CD103⁺ regulatory Tcells or CD4⁺CD25^(high) regulatory T cells in a mammal, comprisingselecting a compound that (i) does not activate or inhibit one or moreof a glucocorticoid receptor, an androgen receptor an estrogenreceptor-α, estrogen receptor-β or a biologically active variant of anyof these biomolecules in human or mammalian cells in vitro by more thanabout 30% when compared to suitable control human or mammalian cells invitro; (ii) has a molecular weight of about 100-1000 Daltons, optionallya molecular weight of about 250-850 Daltons; (iii) when compared to asuitable negative control or normal control, increases or decreases thenumbers or activity of CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁺CD103⁺regulatory T cells, CD4⁺CD25^(high)CD103⁺ regulatory T cells orCD4⁺CD25^(high) regulatory T cells by more than 20% in a suitable assay;and (iv) optionally inhibits or decreases the transcriptional activityor level of NF-κB by about 20-80% in human or mammalian cells in vitrowhen compared to suitable negative control human or mammalian cells invitro; whereby the compound is identified. This method is optionallyconducted using one or more reference compounds such as a compound ofembodiment 16, 17, 18, 19 or elsewhere, e.g., at paragraph 40, 41 or 42,as (1) a reference standard or control or (2) the compound to be testeditself, optionally as compared to a different compound of embodiment16,17, 18, 19 or elsewhere, e.g., at paragraph 40, 41 or 42.

22. The method of embodiment 21 wherein the mammal is a rodent or ahuman.

23. The method of embodiment 21 or 22 wherein the numbers or activity ofCD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁺CD103⁺ regulatory T cells,CD4⁺CD25^(high)CD103⁺ regulatory T cells or CD4⁺CD25^(high) regulatory Tcells are determined by a protocol comprising one, two or three of (a)the method of example 20 or a suitable variation thereof; (b) the methodof example 21 or a suitable variation thereof; or (c) a method in areference cited herein or a suitable variation thereof, wherein the orsuitable variation permits assessment of numbers or activity of theCD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁺CD103⁺ regulatory T cells,CD4⁺CD25^(high)CD103⁺ regulatory T cells or CD4⁺CD25^(high) regulatory Tcells.

The method embodiment 21, 22 or 23 wherein the compound is for thetreatment or prophylaxis of autoimmune disease or unwanted inflammationcondition, which optionally is an arthritis condition such as anosteoarthritis (primary or secondary osteoarthritis), rheumatoidarthritis, an arthritis associated with spondylitis such as ankylosingspondylitis, multiple sclerosis, Alzheimer's disease, tenosynovitis, alupus condition such as systemic lupus erythematosis or discoid lupuserythematosis, tendinitis, bursitis, a lung inflammation condition suchas asthma, emphysema, chronic obstructive pulmonary disease, lungfibrosis, cystic fibrosis, acute or adult respiratory distress syndrome,chronic bronchitis, acute bronchitis, bronchiolitis, bronchiolitisfibrosa obliterans, bronchiolitis obliterans with organizing pneumonia.The compound can be a formula 1 compound as described herein.

25. A method to treat a metabolic disease in a human or a rodent havingthe metabolic disease, or subject to developing the metabolic diseasecomprising administering to the human or the rodent a treatmenteffective amount of a compound, optionally wherein the compound is apartial inhibitor of NF-κB in an in vitro assay or the compound isidentified by the method of claim 1 in this application as originallyfiled or as described elsewhere herein. Such treatments includetreatment with about 0.1 mg/day, about 1 mg/day or about 5 mg/day toabout 40 mg/day or about 80 mg/day of a F1C described herein such as17α-ethynylandrost-5-ene-3β,7β,17β-triol. Other F1C that can be used inthis embodiment include 17α-ethynylandrost-5-ene-3α,7α,17α-triol,17α-ethynylandrost-5-ene-7β,17β-diol-3-one,17α-ethynylandrost-5-ene-3β,17β-diol-7-one,17α-ethynylandrost-5-ene-3α,17β-diol-7-one,17α-chloroethynylandrost-5-ene-3β,7β,17β-triol,17α-chloroethynylandrost-5-ene-3α,7β,17β-triol,androst-5-ene-3β,4β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3β,4β,16α,17β-tetrol,androst-5-ene-3β,4β,7β,17β-tetrol,17α-ethynylandrost-5-ene-3β,4β,7β,17β-tetrol,3β,4β-diacetoxyandrost-5-ene-7β,17β-diol,3β,4β-diacetoxy-17α-ethynylandrost-5-ene-7β,17β-diol,3α,4β-diacetoxyandrost-5-ene-7β,17β-diol,3α,4β-diacetoxy-17α-ethynylandrost-5-ene-7β,17β-diol,androst-5-ene-2β,3α, 7β,17β-tetrol,17α-ethynylandrost-5-ene-2β,3α,7β,17β-tetrol andandrost-5-ene-2α,3β,7β,17β-tetrol,17α-ethynylandrost-5-ene-2α,3β,7β,17β-tetrol and analogs of thesecompounds where a —OH moiety replaces a hydrogen atom at the 18- or19-position. These compounds include17α-ethynylandrost-5-ene-3β,7β,17β,18-tetrol,17α-ethynylandrost-5-ene-3α,7β,17β,18-tetrol,androst-5-ene-3β,4β,7β,17β,18-pentol,17α-ethynylandrost-5-ene-3β,4β,7β,17β,18-pentol,17α-ethynylandrost-5-ene-3β,7β,17β,19-tetrol,17α-ethynylandrost-5-ene-3α, 7β,17β,19-tetrol,androst-5-ene-3β,4β,7β,17β,19-pentol,17α-ethynylandrost-5-ene-3β,4β,7β,17β,19-pentol and compound describedelsewhere. Human subject to developing diabetes are ones that arepre-diabetic and often obese.

26. The method of embodiment 25 wherein the F1C has the structure

wherein one R¹ is —H or C₁₋₈ optionally substituted alkyl and the otherR¹ is —OH, an ester or an ether; one R² is —H or C₁₋₈ optionallysubstituted alkyl and the other R² is —OH or an ester, or both R²together are ═O; one R³ is —H and the other R³ is —H, —OH, an ester oran ether; one R⁴ is optionally substituted C₂₋₄ alkynyl; one R⁴, e.g.,R⁴ in the β-configuration, is —OH, an ester or an ether; R⁷ is —CH₃,—CH₂OH or —C₂H₅; and R¹⁵ is —H, a halogen, e.g., —F, —OH, ═O, an esteror an ether.

27. The method of embodiment 25 or 26 wherein the metabolic disorder istype 2 diabetes, hyperglycemia, elevated nonesterified fatty acids,insulin resistance or another metabolic condition described herein orwherein the compound has the structure

For some of these structures R² is —OH. For other structures, R² is—OC(O)CH₃. For other structures, R² is —OCH₃. For other structures, R²is an ether such as —OC₂H₅ or —OC₃H₇. For other structures, the hydroxylat the 17 is esterified with an ester such as —OC(O)CH₃ (acetate),—OC(O)C₂H₅, —OC(O)—(CH₂)₁₀—CH₃, —OC(O)—(CH₂)₁₄—CH₃, —OC(O)—(CH₂)₁₆—CH₃.

28. A compound having the structure

wherein the dotted line is an optional double bond and if no double bondis present at the 5-6 position, hydrogen is present at the 5-position inthe α- or β-configuration; one R¹ is —H or C₁₋₈ optionally substitutedalkyl and the other R¹ is —OH, an ester or an ether; one R² is —H orC₁₋₈ optionally substituted alkyl and the other R² is —OH, an ester, anether, or both R² together are ═O; one R³ is —H and the other R³ is —H,—OH, an ester or an ether; R⁴ in the α-configuration is optionallysubstituted alkynyl or optionally substituted C₂₋₄ alkynyl; R⁴ in theβ-configuration is —OH, an ester or an ether; R⁷ is C₁₋₄ optionallysubstituted alkyl such as —CH₂OH; and R¹⁵ is —OH, an ester or an etherin the α- or β-configuration.

29. The compound of embodiment 28 wherein the compound has the structure

wherein R⁷ is —CH₃ or —CH₂OH, and R² is —OH or an ester or ether such as—OCH₃. In some embodiments, (A) R² is —OC(O)CH₃ and R⁷ is —CH₂OH (B), R²is —OC(O)CH₃ and R⁷ is —CH₃, (C)R² is —OH and R⁷ is —CH₃, (D) R² is —OHand R⁷ is —C₂H₅. In other embodiments, hydroxyl at the 11-position, ifpresent, is replaced with acetate. In other embodiments, the double bondat the 5-6 position is absent at hydrogen is present in theα-configuration, e.g., 17α-ethynylandrostane-3β,7β, 11β, 17β-tetrol or17α-ethynylandrostane-3α,7β,11β,17β-tetrol. In other embodiments, thedouble bond at the 5-6 position is absent at hydrogen is present in theβ-configuration, e.g., 17α-ethynyl-5β-androstane-3β,7β,11β,17β-tetrol or17α-ethynyl-5β-androstane-3α,7β,11β,17β-tetrol.

Variations and modifications of these embodiments and other portions ofthis disclosure will be apparent to the skilled artisan after a readingthereof. Such variations and modifications are within the scope of thisinvention. The claims in this application or in applications that claimpriority from this application will more particularly describe or definethe invention. All citations or references cited herein are incorporatedherein by reference in their entirety at this location or in additionalparagraphs that follow this paragraph. Other descriptions are found inpending U.S. application Ser. No. 11/941,934, filed Nov. 17, 2007, U.S.provisional application Ser. No. 60/866,395, filed Nov. 17, 2006, U.S.provisional application Ser. No. 60/866,700, filed Nov. 21, 2006, U.S.provisional application Ser. No. 60/868,042, filed Nov. 30, 2006, U.S.provisional application Ser. No. 60/885,003, filed Jan. 15, 2007, U.S.provisional application Ser. No. 60/888,058, filed Feb. 2, 2007, all ofwhich are incorporated herein by reference.

EXAMPLES

The following examples further illustrate the invention and they are notintended to limit it in any way.

Example 1

Treatment of lung inflammation. Three compounds,3β,16α-dihydroxy-17-oxoandrostane, 3α,160,17β-trihydroxyandrostane and3α,16α,177-trihydroxyandrostane were used to treat inflammation in miceessentially as described (D. Auci et al., Ann. New York Acad. Sci.1051:730-742 2005). Five to 8 week old CD1 male mice (Charles River,Calco, Italy) were used for the study. The animals were housed in acontrolled environment and provided with standard rodent chow and water.Animal care was in compliance with applicable regulations on protectionof animals. Mice were allocated into one of the following groups: (1)mice treated with 2% carrageenan-λ in saline (carrageenan-λ treatedcontrol group), (2) mice treated with 0.1 mg, 0.01 mg or 0.001 mg3β,16α-dihydroxy-17-oxoandrostane by subcutaneous (s.c.) injection 24 hand 1 h before carrageenan-λ administration, (3) mice treated with 0.1mg, 0.01 mg or 0.001 mg of 3α,16α,17α-trihydroxyandrostane by s.c.injection 24 and 1 h before carrageenan; (4) mice treated with 0.1 mg,0.01 mg or 0.001 mg 3α,16β,17β-trihydroxyandrostane by s.c. injection 24h and 1 h before carrageenan-λ administration; (5) mice treated withvehicle (0.1% carboxymethylcellulose, 0.9% saline, 2% tween 80, 0.05%phenol) s.c. 24 h and 1 h before carrageenan-λ administration; (6) micetreated with rabbit anti-mouse polyclonal anti-TNF-α antibody (200 μg)given as an intraperitoneal bolus 24 h and 1 h before carrageenan-λadministration (positive control group); and (7) sham-operated mice thatwere not treated with carrageenan-λ. Each group consisted of 10 mice.All treatments were given in a final volume of 100 μL. Lung (pleuralcavity) inflammation was induced as follows. The mice were anaesthetisedwith isoflurane and a skin incision was made at the level of the leftsixth intercostal space. The underlying muscle was dissected and either0.1 mL saline (control) or 0.1 mL saline containing 2% λ-carrageenan wasinjected into the pleural cavity. The carrageenan-λ is a potent inducerof inflammation, which is manifested in this protocol by accumulation offluid and neutrophils in the pleural cavity. The incision was closedwith a suture and the animals were allowed to recover.

At 4 h after the injection of carrageenan-λ, the animals were euthanizedby exposure to CO₂. The chest was carefully opened and the pleuralcavity rinsed with 1 mL of saline solution containing heparin (5 U/mL)and indomethacin (10 μg/mL). The exudate and washing solution wereremoved by aspiration and the total volume measured. Any exudatecontaminated with blood was discarded. The amount of exudate wascalculated by subtracting the injected 1 mL volume from the totalpleural cavity volume that was recovered. The neutrophils in the exudatewere suspended in phosphate-buffer saline and counted with an opticalmicroscope in a Burker's chamber after Trypan Blue staining. The resultswere analysed by one-way ANOVA followed by a Bonferroni post-hoc testfor multiple comparisons. A p-value less than 0.05 was consideredsignificant. For statistical analysis each group was compared to thecontrol group of mice that were challenged with carrageenan-λ andreceived no other treatment.

All of the mice that were challenged with carrageenan-λ and were leftuntreated developed an acute pleurisy, producing turbid exudate andincreased pleural numbers of neutrophils. The increase in volumeexudates and numbers of leukocytes in the pleura of the mice treatedwith the vehicle was similar to that observed in the control mice thatwere challenged with carrageenan-λ and received no treatment. Relativeto these two groups of control mice, animals treated with3β,16α-dihydroxy-17-oxoandrostane showed a significant reduction in thenumber of neutrophils in the pleura the volume of pleural exudates atthe 0.1 mg 0.01 mg doses, while the lower 0.001 mg dose was inactive.The volume of pleural exudate at the 0.1 mg dose in the treated with3β,16α-dihydroxy-17-oxoandrostane was significantly reduced, but not atthe lower 0.01 mg and 0.001 mg doses. Animals treated with3α,16α,17α-trihydroxyandrostane showed a significant reduction in thenumber of neutrophils in the pleura at the 0.1 mg and 0.01 mg doses.Treatment with 3α,16α,17α-trihydroxyandrostane also showed a significantreduction in the number of neutrophils in the pleura at the 0.1 mg and0.01 mg doses. The potency of 3α,16α,17α-trihydroxyandrostane and3α,16β,17α-trihydroxyandrostane were similar to that observed with thepolyclonal anti-TNF-α antibody control, while3β,16α-dihydroxy-17-oxoandrostane was less potent.

The table below describes the number of neutrophils from the treatedanimal groups relative to untreated control animals that were exposed tocarrageenan-λ, but not treated with anything else (negative controlgroup). The neutrophil number for the negative control group was set at100% and other groups were compared to this. The group of animals thatwere treated with anti-TNF-α antibody (positive control group) had 29%of the number of neutrophils the negative control group had, whichindicates that the antibody had an antiinflammatory effect against thecarrageenan-λ exposure. The vehicle control group did not have asignificantly reduced number of neutrophils (91%) compared to thenegative control group, which shows no significant antiinflammatoryeffect due to the vehicle alone.

3β,16α-dihydroxy-17- 3α,16α,17α- 3α,16β,17β- oxoandrostanetrihydroxyandrostane trihydroxyandrostane 0.001 mg 97% 0.001 mg 103% 0.001 mg 95% 0.01 mg 73% 0.01 mg 45% 0.01 mg 50% 0.1 mg 73% 0.1 mg 30%0.1 mg 42%

Other compounds that had statistically significant anti-inflammationactivity in this model were 17α-ethynylandrost-5-ene-3β,7β,17β-triol (1mg and 0.1 mg administered by oral gavage) and17β-aminoandrost-5-ene-3β-ol (40 mg/kg administered by oral gavage,about 0.5 mg/mouse). These compounds were active as compared to groupsof mice that were used as vehicle controls.

Other formula 1 compounds described herein can be used in this manner tocharacterize their relative capacity to treat or ameliorateinflammation. These compounds include 3β,16β,17β-trihydroxyandrostane,3β,16α,17α-trihydroxyandrostane, 3β,16β,17α-trihydroxyandrostane,3β,16β-dihydroxyandrost-5-ene-17-oxime,3β,16α-dihydroxyandrost-5-ene-17-oxime,3α,16α-dihydroxyandrost-5-ene-17-oxime,3β,16α-dihydroxyandrostane-17-oxime and analogs of these compounds that(1) contain a hydroxyl group at the 7-position in the α-configuration orthe β-configuration and/or (2) a double bond at the 5-position or the4-position, and/or (3) an ester, ether, amino acid, carbamate or oxime(═NOH) derivative, conjugate or analog of any of these.

Example 2

Analysis of the immune response. The compound3α,16α,17α-trihydroxyandrostane was found to have biological propertiesthat make the compound superior as an agent to treat an inflammationcondition such as asthma. Specifically, the use of the compound was notaccompanied by a rebound in IL-13, which is a known side effect ofantiinflammatory glucocorticoid compounds such as dexamethasone. TheIL-13 rebound after glucocorticoid makes an asthma patient more prone tohave subsequent acute flare, so an antiinflammatory agent that does notdo this would be advantageous. This lack of an IL-13 rebound wasunexpected.

The capacity of 3α,16α,17α-trihydroxyandrostane to limit eosinophilburden and to reduce key inflammatory mediators (IL-5, IL-13, cysteinylleukotrienes) was observed in the ovalbumin (OVA) sensitized mouse modelof asthma. BALB/c mice were sensitized by intraperitoneal injection withOVA (in alum adjuvant) on days 1, and 12. Airways were challenged withOVA on days 28 and 30 by delivery of OVA to the lung, or with saline. Onday 31, six mice were with saline and 6 mice challenged with OVA weresacrificed and lung tissue was analyzed. The remaining animals weredivided into 6 groups (6 mice per group). Groups of the mice weretreated once daily by subcutaneous injection as follows. Group 1 vehiclecontrol (0.1% carboxymethyl cellulose, 0.9% saline, 2% tween 80, 0.05%phenol). Group 2 dexamethazone (5 mg/kg). Group 33α,16α,17α-trihydroxyandrostane (1 mg/mouse). Three animals in groups1-3 were sacrificed on day 35 at 1 hr after final treatment and theremaining 3 animals in groups 1-3 were sacrificed on day 38.

As shown in the table below, the 3α,16α, 17α-trihydroxyandrostane didnot generate an IL-13 increase that was observed with animals that hadbeen treated with dexamethasone.

Treatment IL-13 (pg/mL) saline control 220 ovalbumin 230 vehicle (day35) 220 dexamethasone (day 35) 340 3α,16α,17α-trihydroxyandrostane (day35) 195 vehicle (day 38) 190 dexamethasone (day 38) 3903α,16α,17α-trihydroxyandrostane (day 38) 210

In addition to a reduction in the day 38 IL-13 rebound after challenge,the animals treated with 3α,16α,17α-trihydroxyandrostane had a reducedlevel of IL-5 in lung tissue (90 pg/mL) compared to the dexamethasonetreated group (145 pg/mL). The IL-5 level in the vehicle control groupwas 75 pg/mL at day 38. Other formula 1 compounds described herein wereused in this manner to identify their capacity to treat or ameliorateinflammation without an IL-13 and/or IL-5 rebound effect, including3β,16β,17β-trihydroxyandrostane, 3β,16α,17α-trihydroxyandrostane,3β,16β,17β-trihydroxyandrostane, androst-5-ene-2α,3β,16α,17β-tetrolandrost-5-ene-3β,7β,16α,17β-tetrol and17α-ethynylandrost-5-ene-3β,7β,17β-triol. These results show that theF1Cs can be used to treat lung inflammation or asthma in vivo.

In another protocol, a population of mast cells was cultivated frommurine bone marrow as follows. Briefly, bone marrows from Balb/C micewere flushed from the femur using PBS and a 27 g needle. The cells werecultured in a mixture of ⅔ RPMI-1640 with 19% FBS and cells thatsecreted IL-3. The bone marrow cells were allowed to differentiate for18-25 days in the IL-3-containing mixture before being used forexperiments. Bone marrow cells cultured in this manner have a phenotypesimilar to mucosal mast cells and are referred to as bone marrow-derivedmast cells (BMMC).

The homogeneity of the in vitro propagated mast cells was checked byconventional flow cytometry techniques and staining for cell-typespecific markers. Between days 14 and 21 of propagation, mature mastcells were harvested and prepared for the test cultures. The objectivewas to assess of the effect of compounds such as dehydroepiandrosteroneon mast cell stimulus-coupled degranulation. Prepared mast cells weredispensed into test culture wells at a density of 1×10⁷ cells/mL. Incontrol cultures, mast cells were induced to degranulate after crosslinking of IgE receptors with IgE antigen-antibody complexes. Inparallel groups of cultures mast cells were preincubateddehydroepiandrosterone at various doses followed by activation usinganti-IgE antibody. There was no detectable degranulation of mast cellsas measured by release of β-glucuronidase from cytosolic storagegranules of the cells in the absence of the stimulus. Introduction ofanti-Ig-E receptor antibody to the cultures caused a significant releaseof β-glucuronidase. When mast cells were exposed todehydroepiandrosterone alone, there was no measurable degranulation.However, mast cells pre-exposed to doses of 100 μMdehydroepiandrosterone for 5 to 10 minutes before activation withanti-IgE antigen-antibody complexes, exhibited approximately 70%inhibition of degranulation. Lower levels of dehydroepiandrosteroneshowed proportionately less capacity to inhibit degranulation. Insimilar protocols, F1Cs such as17α-ethynylandrost-5-ene-3β,7β,17β-triol,androst-5-ene-3β,7β,16α,17β-tetrol or androst-5-ene-3α,7β,16α,17β-tetrolwere 10-1000 fold more potent than dehydroepiandrosterone.

Example 3

Treatment of lethal inflammation/shock. Two compounds,16α-bromoepiandrosterone (3β-hydroxy-16α-bromoandrostane-17-one) and3β,16α-dihydroxy-17-oxoandrostane, were used in a lethal shock protocol.In one protocol, 3 mg of 16α-bromoepiandrosterone was administered toone group of animals by oral gavage, while another group received 3 mgof 16α-bromoepiandrosterone by subcutaneous injection. A group ofcontrol animals received a placebo control. In this protocol, the16α-bromoepiandrosterone was administered to mice at 24 hours before andat 1 hour after administration of a lethal amount of bacteriallipopolysaccharide (LPS). By the end of the observation period, 72 hoursafter LPS administration, none of the vehicle treated placebo controlanimals had survived, while 65% of animals that received16α-bromoepiandrosterone by oral administration survived. 50% of theanimals that received 16α-bromoepiandrosterone by subcutaneous injectionsurvived. Animals that survived for 72 hours all recovered from the LPSexposure.

In a second assay, 16α-bromoepiandrosterone or3β,16α-dihydroxy-17-oxoandrostane was administered to mice by oralgavage at 24 hours before and 1 hour after administration of a lethalamount of LPS. A vehicle treated group of animals was used as theplacebo control. At 72 hours, 25% of the placebo control mice survived,50% of the mice treated with 3β,16α-dihydroxy-17-oxoandrostane survivedand 80% of the mice treated with 16α-bromoepiandrosterone survived.

In another assay, the capacity of 16α-bromoepiandrosterone and3β,16α-dihydroxy-17-oxoandrostane to protect against lung injury inducedby exposure to a sublethal amount of LPS in mice was shown. In thisassay, the compounds, sterile saline (negative control) or vehicle(vehicle control) were administered to groups of 5 mice by oral gavageat 24 hours before and 1 hour after administration of 100 μg of LPS tothe trachea and lungs of animals under light anesthesia. At 48 hours theanimals were sacrificed and samples were obtained from the lungs of theanimals by bronchiolar alveolar lavage (BAL). The numbers of cells inthe BAL fluid were counted, with high numbers of cells showing lunginflammation and damage. In this assay, cells that mediate inflammationand lung damage infiltrate into the lungs in response to the presence ofthe LPS. In the negative control and vehicle control groups, the BALfluid contained about 6×10⁷ cells/mL. The numbers of cells in the groupsof animals that were treated with 16α-bromoepiandrosterone (p=0.02) or3β,16α-dihydroxy-17-oxoandrostane (p=0.04) had significantly reducedcell counts in the BAL fluid (about 4.4×10⁷ cells/mL). This result showsthe compounds may have activity in clinical conditions such as asthma orCOPD where lung injury or damage is associated with uncontrolled orexcess inflammation. Other compounds, e.g.,17α-ethynylandrost-5-ene-3β,7β,17β-triol or17β-aminoandrost-5-ene-3β-ol, can be characterized in a similar manner.

Example 4

Clearance of bacteria from lung tissue. The capacity of16α-bromoepiandrosterone to clear a Pseudomonas aeruginosa infectionfrom lung tissue was shown using a previously published protocol, A. vanHeeckeren et al., J. Clin. Invest., 100(11):2810-2815 1977; A. vanHeeckeren et al., Am. J. Respir. Crit. Care Med., 161:271-279 2000. Theprotocol was conducted in CFTR mice, which are used as an animal modelfor human cystic fibrosis, S. D. Freedman et al., Proc. Natl. Acad. Sci.USA, 96(24):13995-14000 1999; W. Zeng et al., Am. J. Physiol. Cell.Physiol. 273:C442-C455 1997. Establishment of chronic P. aeruginosainfection using agarose beads containing bacteria (50 μL containingabout 6.1×10⁴ CFU/animal) was published earlier, J. R. Starke et al.,Pediatr. Res., 22:698-702 1987. Two groups of mice (n=9 for each group)were treated with 40 mg/kg of 16α-bromoepiandrosterone or vehicle(control) and the bacterial burden in the lungs of the animals wasdetermined at 10 days after introduction of the agarose beads into thelung. At day 10, the bacterial burden in the lungs of the vehiclecontrol animals was about 6×10⁶ CFU/animal, while the animals treatedwith 16α-bromoepiandrosterone had a reduced (p=<0.04) bacteria burden.This result shows that 16α-bromoepiandrosterone can be used to treat orreduce lung infection, which is a desirable attribute for agents thatare used to treat conditions such as cystic fibrosis.

Example 5

Anti-inflammation activity in human cells in vitro. The capacity of16α-bromoepiandrosterone and 3β,16α-dihydroxy-17-oxoandrostane to reduceinflammation in human cells in vitro was demonstrated using human wholeblood that was exposed to LPS. Reduced production of γ-interferon by thecells was observed in the presence of 16α-bromoepiandrosterone (100mg/mL) and 3β,16α-dihydroxy-17-oxoandrostane compared to cells exposedto LPS alone (positive control) or vehicle (dimethylsulfoxide) withoutcompound (vehicle control). The amount of γ-interferon was measured inthe growth medium when the cells had been incubated in the presence ofLPS for 24 hours.

Example 6

Treatment of autoimmune neurodegeneration. Three compounds,17β-aminoandrost-5-ene-3β-ol, 17β-dimethylaminoandrost-5-ene-3β-ol and17β-methylaminoandrost-5-ene-3β-ol were characterized for their capacityto ameliorate experimental allergic encephalomyelitis (EAE) in mice.This demyelinating condition is extensively used as a model for multiplesclerosis in humans and for testing of new therapies for treatingmultiple sclerosis, e.g., B. F. Bebo Jr. et al., J. Neurosci. Res.52:420-426 1998; R. R. Voskuhl et al., Neuroscientist, 7:258-270 2001;H. Offner et al., J. Neuroimmunol., 130:128-139 2002. Activity in thismodel shows the capacity of test compounds to prevent or slow the rateof neuron death that is associated with progression of the EAE disease.

In this protocol, the compounds were administered to female SJL/J miceby oral gavage at the onset of disease symptoms. An antigen was used toinitiate the EAE condition in the mice. The antigen that was used forthe active immunization was mouse proteolipid protein (PLP) residues139-151. Immunization with this peptide antigen initiates an autoimmuneTh1 mediated demyelinating disease of the central nervous system. Theantigen was prepared by solid phase synthesis and purified byhigh-performance liquid chromatography. The EAE condition was initiatedin the female SJL/J mice by immunization with 150 μg of the PLP 139-151peptide in complete Freund's adjuvant containing 200 μg of Mycobacteriumtuberculosis. The immunization protocol was subcutaneous injection overfour sites on the hind flank on day 0. After immunization. the mice wereassessed daily for clinical signs of EAE using the following scale: 0=noclinical signs or symptoms; 1=limp tail; 2=mild hind limb weakness andlimp tail; 3=moderate hind limb weakness and limp tail or mild ataxia;4=severe hind limb weakness and mild forearm weakness with moderateataxia; 5=paraplegia with no more than moderate forelimb weakness;6=paraplegia with severe forelimb weakness or severe ataxia or moribundcondition.

Mice in the vehicle control group began to show observable symptoms ofEAE at about 10-11 days after immunization with the PLP antigen, whichis typical for the EAE disease model. The animals were dosed daily with17β-aminoandrost-5-ene-3β-ol, 17β-dimethylaminoandrost-5-ene-3β-ol or17β-methylaminoandrost-5-ene-3β-ol by oral gavage beginning at day 1,which was 1 day after immunization. All three of the compounds wereactive at a dose of 5 mg/kg and they reduced the clinical severity ofthe symptoms that were observed through day 26, when the observationperiod ended. The therapeutic activity for the compounds was observed atblood levels of about 10 mg/mL in the mice. These results showed thatthe compounds were biologically active in treating this chronicautoimmune neurodegeneration disease.

Example 7

Inhibition of NF-κB in vitro. A number of compounds were used to inhibitactivation of NF-κB by TNF-α or LPS in human cells in vitro. Activationof NF-κB increases expression of a number of genes that mediateinflammation. This protocol used human THP-1 cells, which are humanmononuclear blood cells with a monocyte phenotype. The cell line,referred to as NF-κB-bla THP-1, contained a β-lactamase reporter geneunder the control of the NF-kB response element (Invitrogen,CellSensor™, product No. K1176). In this cell line, the β-lactamasereporter gene is stably integrated in the THP-1 cells. This cell linewas used to detect agonists or antagonists of the NF-κB signalingpathway. These NF-κB-bla THP-1 cells respond to the presence of tumornecrosis factor alpha (TNFα) or bacterial lipopolysaccharide (LPS) byincreased expression of the β-lactamase reporter gene. The level ofβ-lactamase enzyme activity was measured by fluorescence resonanceenergy transfer ratiometric detection. TNFα and LPS are both potentinflammation-inducing agents that activate NF-κB in THP1 cells. In thisassay, compounds that decrease NF-κB activity, and thus β-lactamase, inthe presence of TNFα or LPS are exerting an anti-inflammation activity.

The NF-κB-bla THP-1 cells were maintained by passaging or feeding asneeded. The cells, which grow in suspension, were maintained at adensity between 2×10⁵ cells per mL and 2×10⁶ cells/mL. The cells wereplated at 20,000 cells/well in a 384-well Black-wall, clear bottom assayplates (Costar#3712-TC low fluorescence background plates) approximately24 hours before adding either TNFα at 10 mg/mL or LPS at 0.2 mg/mL toactivate NF-κB. In positive control assays for activation of NF-κB, theEC₅₀ concentration for TNFα was 0.20 mg/mL after a 1 hour β-lactamasesubstrate incubation. The EC₅₀ dose for LPS was 0.15 mg/mL. The EC₅₀concentration for TNF-α or LPS in this assay refers to 50% of theconcentration of TNF-α or LPS that causes a maximum activation of NF-κB.The synthetic glucocorticoid dexamethasone (a potent anti-inflammatorydrug) decreased the effect of TNFα by with an EC₅₀ of 0.47 nM (averageof 5 assays) in this assay. Similar biological activity fordexamethasone has been reported in other in vitro cell assays, withcomplete inhibition of NF-kB activation observed at an IC₅₀ of about 1nM (M. K. A. Bauer et al., Eur. J. Biochem. 243:726-731, 1977).

Using this assay, the IC₅₀ of compounds for inhibition of NF-κBactivation in NF-κB-bla THP-1 cells after LPS stimulation is shownbelow. The IC₅₀ concentration for the compounds used in this assayrefers to the concentration of compound that causes a 50% of the maximuminhibition of NF-κB activation that the compound can induce. The assayswere usually conducted 2-4 times for each compound and the values shownbelow are averages for each compound. The data in Table 1 below showsthat very low levels of many of these compounds can inhibit NF-κB inthese human macrophage cells.

TABLE 1 IC₅₀* compound 0.47 nM ± 0.11 dexamethasone (positiveanti-inflammation control)  >10 μM estradiol (negative anti-inflammationcontrol)  8.2 fM ± 7.4 3β,7β,16α,17β-tetrahydroxyandrost-5-ene 84.5 fM ±65 3α,7β,16α,17β-tetrahydroxyandrost-5-ene  >10 μM3β,7α,16α,17β-tetrahydroxyandrost-5-ene  >10 μM16α-acetoxy-3β,7β,17β-trihydroxyandrost-5-ene  0.4 fM3β,4β,16α,17β-tetrahydroxyandrost-5-ene 0.01 fM4β-acetoxy-3β,16β,17β-trihydroxyandrost-5-ene  2.0 fM3β-acetoxy-7β,11β,17β-trihydroxyandrost-5-ene  >10 μM3β,7β,11β,17β-tetrahydroxyandrost-5-ene   10 fM3β,7β,17β-trihydroxy-11-oxoandrost-5-ene  0.1 pM17α-methyl-3β,11α,17β-trihydroxyandrost-5-ene  >10 μM3β,11α-dihydroxy-17-oxoandrost-5-ene  2.0 fM2α,3β,17β-trihydroxyandrostane   14 pM ± 123β,17β-dihydroxyandrost-5-ene  1.2 fM ± 0.283β,7β,17β-trihydroxyandrost-5-ene  >10 μM3β,7α,17β-trihydroxyandrost-5-ene   19 fM ± 113β,7β,17β-trihydroxy-17α-ethynylandrost-5-ene  >10 μM3β,7β,17β-trihydroxy-17α-trifluoromethylandrost-5-ene  6.8 fM ± 5.63β,7α,17β-trihydroxy-17α-ethynylandrost-5-ene   12 fM ± 9.83β,7β,17β-trihydroxy-17α-vinylandrost-5-ene 50.3 fM ± 13.93β,7β,17β-trihydroxy-17α-methylandrost-5-ene   64 fM ± 363β,7α,17β-trihydroxy-17α-methylandrost-5-ene   30 pM ± 2916α-fluoroandrost-5-ene-17-one  1.9 nM ± 0.8 16α-iodoepiandrosterone 8.8 μM ± 1.3 16α-bromoepiandrosterone  0.6 μM ± 0.216β-bromoepiandrosterone  >10 μM 16α-hydroxyepiandrosterone  7.2 fM ±4.7 3β,17β-dihydroxy-17α-methylandrost-5-ene 11.5 fM ± 3.53β,17β-dihydroxy-7-oxo-17α-ethynylandrost-5-ene  >10 μM3β,17β-dihydroxy-7-oxo-17α-methylandrost-5-ene *μM = 10⁻⁶ M; nM = 10⁻⁹M; pM = 10⁻¹² M; fM = 10⁻¹⁵ M

Other compounds that showed anti-inflammatory activity in this protocolwere 3α-pentafluoroethylandrost-4-ene-3β,17β-diol (IC₅₀ 3.1 nM),3α-pentafluoroethylandrost-5-ene-3β,17β-diol (IC₅₀ 17 nM; maximum NF-κBinhibition was 50%), 3α/β, 17α-ethynylandrostane-3α/β, 17β-diol (IC₅₀200 pM), 17α-trifluoromethylandrostane-3α,17β-diol (IC₅₀ 190 nM),17β-glycylandrostane-3β-ol (IC₅₀ 0.42 pM), 3β-glycylandrostane-17β-ol(IC₅₀ 1 nM), androstane-3β,16β-diol-17-oxime (IC₅₀ 1.9 fM)17α-ethynylandrost-4-ene-3-one-17β-ol (IC₅₀ 2.9 fM; maximum NF-κBinhibition was 80%), 16α-fluoroandrost-5-ene-17-one (IC₅₀ 30 pM),16β-fluoroandrost-5-ene-7β-ol-17-one (IC₅₀ 1.5 nM),androstane-3α,16α,17β-triol (IC₅₀ 6.9 fM), androstane-3α,16β,17β-triol(IC₅₀ 19 fM), androst-5-ene-3β-ol-17β-succinyl ester (IC₅₀ 0.2 nM),3β-acetoxy-7β,17β-dihydroxy-11-oxoandrost-5-ene (IC₅₀ 1 fM; maximumNF-κB inhibition was 65%). Maximum inhibition of NF-κB by thesecompounds was about 25% to 80%, which differed from 100% inhibition ofNF-κB activation by the synthetic glucocorticoid dexamethasone in thisprotocol.

Two compounds increased NF-κB activity in this protocol,androst-5-ene-3β,7α,16α-triol-17-one (IC₅₀ 1.3 nM; 140% NF-κB activitycompared to control cells) and 3β,17α-dimethylandrostane-3α,17β-diol(IC₅₀ 40 nM).

Compounds that did not exhibit anti-inflammation activity in thisprotocol were 3β,17α-methylandrostane-3β,17β-diol,3β-acetoxyandrost-5-ene-3β,17β-diol,17α-methylandrost-5-ene-3β,17β-diol-7-one,16α-fluoroandrost-5-ene-7β-ol-17-one,16α-fluoroandrost-5-ene-7α-ol-17-one,17α-methylandrostane-3β,7α,17β-triol, androst-5-ene-3β, 11β,17β-triol,16α-fluoroandrostane-17-one, androst-5-ene-3α,17β-diol,androstane-2β,3α,16α, 17β-tetrol and androstane-3α,16α,17α-triol, all ofwhich had an IC₅₀>10 μM.

The capacity of the compounds to decrease the activity of NF-κB at lowlevels indicates that they can be used to treat inflammation,particularly in conditions where excess levels or nuclear transcriptionactivity mediated by NF-kB plays a significant role in the pathology ofthe disease or condition.

In the assay described above, maximum inhibition of NF-κB bydexamethasone, 16α-bromoepiandrosterone and 16β-bromoepiandrosterone was100% and there was no detectable NF-κB activation at concentrations ofthese compounds above the IC₅₀ for these compounds. By contrast, maximuminhibition of NF-κB by the other compounds e.g.,3β,7β,16α,17β-tetrahydroxyandrost-5-ene, 3α,7β,16α,17β-tetrahydroxyandrost-5-ene or3β,7β,17β-trihydroxy-17α-methylandrost-5-ene was less than about 80%,with increasing amounts of the compounds above their IC₅₀ levels notproviding significant additional inhibitory activity against NK-κBactivation.

Several compounds in Table 1 had no detectable capacity to exert ananti-inflammation activity in the in vitro cell assay. Other compoundsthat were tested and had no activity in the assay (IC₅₀>10 μM) included3β,17α-dihydroxyandrost-5-ene, dehydroepiandrosterone(3β-hydroxyandrost-5-ene-17-one), 3β-hydroxyandrostane-7,17-dione,16α-bromo-3β,17β-dihydroxyandrost-5-ene and16β-bromo-3β-hydroxyandrost-5-ene-17-one. Nonetheless, some of thosecompounds that were inactive in this in vitro cell assay, e.g.,16α-hydroxyepiandrosterone, were found nonetheless to beanti-inflammatory in animals in vivo. This result shows that thecompounds may act through different mechanisms or that their activityrequires more than cells from a single cell line.

Methods to modulate NF-κB that have been described and that can beincorporated into or used in the practice of the present inventioninclude those described in the following publications. U.S. Pat. Nos.5,989,835, 6,410,516, 6,545,027, 6,831,065 and 6,998,383. Other aspectsof NF-κB activity have been described and can also be incorporated intothe invention methods, e.g., A. S. Baldwin, Annual Rev. Immunol.14:649-683 1996; M. Muller et al., Mol. Cell. Biol. 22((4)1060-10722002; P. A. Baeuerle, Cell 195:729-731 1998.

Example 8

The capacity of selected compounds to treat LPS inducedshock/inflammation in mice was examined by a protocol similar to theprotocol described above. Five groups of three ICR mice weighing about30 g were each treated by intraperitoneal injection with 120 μL vehicle(30% sulfobutylether-cyclodextrin in water),androst-5-ene-3α,7β,16α,17β-tetrol in vehicle,androst-5-ene-3β,4β,16α,17β-tetrol in vehicle or4β-acetoxyandrost-5-ene-3β,16α,17β-triol in vehicle. All drug andvehicle formulations were solutions, not suspensions. Thesulfobutylether-cyclodextrin was obtained commercially (Captisol™,www.cyclexinc.com). There were two vehicle control groups one groupreceived vehicle alone and the other received vehicle plus LPS. Thevehicle or drug was administered 24 hours before and at 1 hour after LPS(about an LD_(50/24) dose, i.e., 50% lethal at 24 hours after LPSadministration) was administered to the mice by intraperitonealinjection. Drug was administered at about 40 mg/kg (1.2 mg drug/animalfor each administration of the drugs). Spleens were harvested from theanimals at 1.5 hours after injection of LPS and spleen cells were lysedand assayed for activated NF-κB by isolating nuclei from spleen cellsand measuring NF-κB from the lysed nuclei. The results indicated thatall three compounds decreased the level of NF-κB activation compared tothe LPS+vehicle control group by about 50%. The level of activated NF-κBin spleen cells from the animals that were treated with vehicle and noLPS, was essentially the same as the activated NF-κB in spleen cellsfrom drug treated animals. These results indicated a potentanti-inflammation effect in the animals as shown by a decrease inactivated NF-κB in drug treated animals compared to control animals.

Example 9

Kinetic analysis of NF-kB inhibition in vivo. The kinetics of NF-kBinhibition after injection of bacterial LPS in mice was examined tofurther probe the mechanism of action of compounds such as17α-ethynylandrost-5-ene-3β,7β,17β-triol, which will only partiallyinhibit activation of NF-κB that is induced by LPS or TNFα in immunecells (macrophages or monocytes) in vitro as described in example 7. Inthis study, mice were treated with17α-ethynylandrost-5-ene-3β,7β,17β-triol (about 40 mg/kg, about 1.2mg/animal) by intraperitoneal injection of a solution (not a suspension)of the compound in the vehicle described in example 8. The drug wasinjected 24 hours before intraperitoneal injection of bacterial LPS(about an LD_(50/24)). The study used two groups of 12 animals, vehiclecontrol or drug administered 24 hours before LPS challenge. Spleens wereharvested from 3 animals from both groups just before LPS challenge andat 1.5, 2.0 and 2.5 hours after administration of LPS. Spleen cells wereharvested and the level of activated NF-κB was measured by assay ofNF-κB in nuclei essentially as described in example 8.

Maximum NF-κB activation after LPS administration occurred at 1.5 hoursin the vehicle controls, which was 4-fold increased over the pre-LPSlevel of activated NF-κB. The results are shown below. The values forthe vehicle control and drug treated animals are relative opticaldensity units from ELISA measurement of NF-κB in nuclei from spleencells.

Time vehicle drug (hours) control treated 0 18 22 1.5 72 2 2.0 10 7 2.510 9

The profound inhibition of NF-κB at the 1.5 hour time point andrelatively normal levels of NF-κB activity at the other time pointsindicated that the compound exerted a transient but potent inhibition ofLPS induced trauma at a critical period after LPS exposure. Similarassays in other studies showed that the level of activated NF-κB at 30minutes and 60 minutes after injection of LPS in vehicle control micewas similar to the pre-LPS time point in this study. This resultindicates that in this model, the effect of LPS on the activation ofNF-κB in spleen cells is maximal at about 1.5 hours post LPS challenge.This time point reveals a convenient time or window at which theactivity of drug candidates can be assessed in vivo, i.e., at about 75minutes to about 105 minutes after LPS challenge. A component of thebeneficial biological activity of such drug candidates can includemoderation or reduction of inflammation that is at least transient,e.g., lasting for about 15 minutes or 30 minutes 45 minutes or more. Thewindow can vary, depending on the route of administration of thebiological insult, e.g., LPS or TNFα, administered by intraperitonealinjection versus LPS or TNFα administered by subcutaneous orintramuscular injection.

Analysis of LPS induced TNFα expression in mice showed that TNFα levelspeaked at 1.5 hours after LPS challenge (500 μg of LPS administered byintraperitoneal injection) with highest levels of TNFα observed at 1-2hours after LPS challenge. TNFα levels at 30 minutes after LPS and at2.5 hours were lower.

Other compounds that can be analyzed for their capacity to act asbiodynamic drugs include androst-5-ene-3β,7β,16α,17β-tetrol,androst-5-ene-3α,70,16α,17β-tetrol, androst-5-ene-3β,7α,16α,17β-tetrol,androst-5-ene-3β,4β,16α,17β-tetrol, androst-5-ene-3β,4α,16α,17β-tetrol,androst-5-ene-3α,4β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3β,7α,17β-triol,17α-ethynylandrost-5-ene-3β,7α,17β-triol,17α-ethynylandrost-5-ene-3β,17β-triol-7-one and pharmaceuticallyacceptable analogs of any of these compounds, e.g., analogs that arehydroxyl ester or ether derivatives at 1, 2 or more hydroxyl groups.Suitable esters and ethers include acetate, n-propionate, i-propionate,succinate, —O—C(O)—(CH₂)_(n)—CH₂R, —O—C(O)—O—(CH₂)_(n)—CH₂R,—O—C(O)—NH—(CH₂)_(n)—CH₂R, amino acid such as glycine and alanine(—O—C(O)—CHCH₃—COOH), hydroxy esters and methyl, ethyl, n-propyl,i-propyl —O—(CH₂)_(n)—CH₂R, —O—(CH₂)_(n)—O—CH₂R (e.g., —O—CH₂CH₂—O—CH₃)ethers, wherein n is 1, 2, 3, 4, 5 or 6 and R is —H, —F, —Cl, —Br, —I,—OH, —C(O)OH (or an acceptable salt, e.g., sodium or potassium salt),—C(O)OCH₃, —C(O)OC₂H₅.

Example 10

The capacity of formula 1 compounds to affect the course of arthritis ina passive collagen induced arthritis model of arthritis was examinedessentially as previously described (E. Simelyte et al., Arthritis &Rheumatism, 52(6):1876-1884, 2005; Z. Han et al. Arthritis & Rheumatism46(3):818-823, 2002; H. Miyahara et al. Clin. Immunol. Immunopathol.,69(1):69-76, 1993). In this protocol, passive collagen-induced arthritiswas induced in DBA/1 mice by administering anti-type II collagenantibodies, which induced an immune response against joint tissue in theanimals. Efficacy in this model of arthritis shows efficacy primarilyagainst inflammation, which is assessed in isolation from cellulareffects that operate in arthritis. The severity of arthritis wasassessed using a semiquantitative clinical scoring system. Groups of 8animals per group were treated with17α-ethynylandrost-5-ene-3β,7β,17β-triol at 40 mg/kg/day for 14 days orvehicle for 14 days by oral gavage. The vehicle was 30%cyclodextrin-sulfobutylether in water and the drug solution was vehiclewith drug at 20 mg/mL.

The animals were examined by measuring ankle thickness and arthritisscore (4-point/paw) with a higher score indicating a more severearthritis. The experiment was terminated after about 14 days, andhistology and gene expression measurements were performed. Forhistology, the left hind paw was harvested, fixed in 10% formalin for 24h, decalcified, and embedded in paraffin. Tissue sections were stainedwith hematoxylin and eosin for safranin O-fast green to determineproteoglycan content. A semi-quantitative scoring system was used toaccess synovial inflammation, extraarticular inflammation, erosion andproteogylcan loss.

Treatment with the compound began following administration of theantibodies. The protocol allowed observation of the effects of treatmenton the progression of arthritis. The results showed that collageninduced arthritis in group 1 was reduced in group 1 animals compared togroup 4 animals and at days 7-14. The maximum clinical score in vehicletreated animals was 10.2 at day 8 compared to a maximum clinical scoreof 5.1 in group 1 animals at day 7. At the end of the protocol at day14, the vehicle treated group clinical score was 7.8 compared to thecontrol group score, which was 4.1. Differences in clinical score atdays 7-14 were apparent in the treated animals, which showed a reducedlevel of inflammation was present in the treated animals compared to thevehicle control animal group. The effect of treatment with17α-ethynylandrost-5-ene-3β,7β,17β-triol was similar to treatment withdexamethasone, which also inhibits inflammation and reduces the severityof arthritis in this animal model. The capacity of17α-ethynylandrost-5-ene-3β,7β,17β-triol to reduce the severity ofarthritis contrasts with suppressors of cell mediated immunity such asmethotrexate or anti-TNFα agents, which have little efficacy in thisarthritis model.

Example 11

The capacity of formula 1 compounds to affect LPS-induced lung injury inthe mouse was investigated. LPS-induced lung injury models previouslyhave been used to evaluate treatments for acute lung injury (ALI), acuteadult respiratory distress syndrome (ARDS) and endotoxin shock or sepsis(Metz et al., C., Chest 100(4): 1110-9,1991; Windsor, A. C. et al., Ann.J. Med. Sci. 306(2): 111-6, 1993; Brigham K. L. et al., Am. Rev. Respir.Dis. 133(5): 913-27, 1986).

The protocol conducted was essentially as described in Su, X. et al.,Intenstive Care Med. 30:133-140, 2004. Female mice 6-8 week old C57/BL6mice (average body weight of 25 g) obtained from Jackson Laboratory (BarHarbor, Me.) were randomized into groups of seven animals and weremaintained under standard housing and food. The groups were (1) micetreated with saline and LPS, (2) mice treated with vehicle and LPS (3)mice treated with 125 μg dexamethasone, (4) mice treated with 40 mg/Kgandrost-5-ene-3β,7β,16α,17β-tetrol and LPS, (5) mice treated with 40mg/Kg 5α-androstane-3β,16α-diol-17-one and LPS, (6) mice treated with 40mg/Kg 5α-androstane-3β,17β-dihydroxy-16-oxime and (7) mice treated with40 mg/Kg androst-5-ene-3α, 7β,16α,17β-tetrol.

On day −1 mice were pre-treated with compound or vehicle. On day 0 micewere treated with a second dose of compound or vehicle. On day 0+60minutes, mice were challenged with 100 μg of E. Coli LPS (Sigma) underdirect visualization of the trachea under light anesthesia. On day 2(i.e. 48 hour time point after LPS challenge) mice were sacrificed miceand BAL obtained (where cell counts and TNFα/IL6 levels were measured).The lungs were taken, minced and used for myeloperoxidase (MPO) studies.LPS-induced acute lung inflammation was preformed by instilling 50 mgLPS (E. Coli 0111:B4, Sigma-Aldrich) in 100 mL PBS into the tracheas oflightly anesthetized (isoflurane) under direct visualization. At 48 htime point, the mice were sacrificed. After this, a tracheotomy isestablished after exposing the trachea in the lower neck. A blunt ended20 gauge needle is inserted into the exposed trachea, which is then tiedoff and used to obtain the bronchoalveolar lavage (BAL). To minimizeairway bleeding and trauma, BAL is performed using 0.5 mL of sterile PBSX 3. A total of 1300 mL are typically recovered from this process. Celldifferential leukocyte counts are determined in BAL fluid (BALF) using ahemacytometer. Differentials are performed on 80-100 cells. Afterobtaining the BAL, the chest cavity is opened and the heart/lungs areperfused with 3 mL of sterile saline through a R ventricular puncture.All of the lung tissue is then harvested and prepared for the MPO assay.For this assay, lungs are individually homogenized in potassiumphosphate buffer (pH 6.0 containing 0.5% hexadecyltrimethylammoniumbromide). Following centrifugation (14,000×g, 10 min 4° C.) 50 μL ofsupernatant was added to 950 μL potassium phosphate buffer containing0.2 mg/mL o-dianisidine dihydrochloride (Sigma-Aldrich) and 0.00002%hydrogen peroxide. Changes in absorbance are measured at 460 ηm.Cytokine levels are determined in BALF cell-free supernatant (200×g, 10min, 4° C.) by ELISAs for TNFα, IL-6 (R&D Systems) using commerciallyavailable ELISAs. Particularly striking are the results forandrsost-5-ene-3β,7β,16α,17β-tetrol for which it was found that animalstreated orally with this compound had reduced levels of MPO, TNFα andIL-6 in BAL as compared to vehicle treated animals. The effect on MPO,which is a measure of neutrophil burden in the lung, and thepro-inflammatory cytokine TNFα was particularly profound. This suggeststhe ability of the compound to block the migration of pro-inflammatorycells into inflamed tissue as well as to reduce the pro-inflammatorycytokine signaling. In this model, acute inflammation is presumablydriven by LPS stimulation of elements of innate immunity. Many of thesesame mediators are increased and thought to be involved in lunginflammation associated with several disorders, including cysticfibrosis, chronic obstructive pulmonary diseases, acute and chronicbronchitis, and even certain infectious diseases like tuberculosis. Theobservation that treatment with andrsost-5-ene-3β,7β,16α,17β-tetroldramatically reduced MPO and pro-inflammatory cytokine levels in BALF at48 h is in keeping with the anti-inflammatory activities reported hereinfor andrsost-5-ene-3β,7β,16α,17β-tetrol in disease specific models ofchronic inflammation, including EAE.

Example 12

Human mixed lymphocyte reaction (MLR). The capacity of3β,16α-dihydroxy-17-oxoandrostane, 3β,17β-dihydroxy-16-oxoandrostane,17α-ethynylandrost-5-ene-3β,7β,17β-triol, and17β-aminoandrost-5-ene-3β-ol to affect antigen specific stimulation inwhich human T lymphocytes respond to a specific foreign antigen (majorhistocompatibility complex). The MLR is used as an in vitro model ofdelayed type hypersensitivity responses and shows the effect that acompound can have on human antigen-specific T cell responses in vivo.Inhibition of the MLR by a compound shows an immune suppression effectof the compound on lymphocytes. Compounds that do not inhibit the MLRare not immune suppressive for the antigen specific activation ofresponding lymphocytes.

Blood samples were obtained from 3 (2 males, 1 female) fasting, healthyhuman volunteers of 23-31 years old. The subjects did not useimmunomodulatory, anti-allergic drugs or antibiotics in the three monthsbefore the study. The subjects were bled between 9 and 10 AM to limitpossible fluctuations in the circulating levels of hormones or cytokinesthat could have influenced the in vitro responses of their lymphocytes.Peripheral blood mononuclear cells (PBMC) were isolated bycentrifugation on Ficoll-Hypaque (density 1.077, Biochrom AG, Berlin,Germany) gradients and resuspended in culture medium (RPMI 1640supplemented with 2 mM L-glutamine, penicillin (100 U/mL) andstreptomycin (100 mg/mL) (Invitrogen s.rl., Milan, Italy). Autologous(responder) inactivated plasma was used at 10%. Five hundred thousandresponder PBMC (PBMCr) and 500,000 allogeneic irradiated (30 Gy)stimulator PBMC (PBMCs) were mixed at a ratio of 1:1 in 200 μL mediumand cultured for 6 days in flat bottom 96 well plates (Nunc, Roskilde,Denmark) at a concentration of 300 nM or 30 nM for each of the 4compounds. The compounds were dissolved in ethanol and then diluted tothe desired concentration with culture medium leading to a finalsolution containing 0.01% of ethanol. This vehicle was used as control.Controls also included PBMCr and PBMCs cultured separately. During thelast 8 hours of the culture period the PBMC were pulsed with 1 μCi/well[³H] thymidine (Amersham, Milan, Italy). The cells were then harvestedand radioactivity incorporation measured with a beta cell counter. Themean cpm of quadruplicate wells was calculated. Proliferation of T cellswas expressed as a stimulation index: SI=cpm (PMBCs×PBMCr)/cpm(PBMCr)+cpm (PBMCs). Statistical analysis was performed using theStudent's t test. The cpm obtained from quadruplicate of each testcompound were compared to proliferative responses obtained in controlPBMCr and PBMCs cultured in the presence of the vehicle. Differenceswere considered significant at p<0.05.

The results showed no inhibition of the MLR by any of the 4 compoundsexcept 17β-aminoandrost-5-ene-3β-ol at 300 nM. This indicated that3β,16α-dihydroxy-17-oxoandrostane, 3β,17β-dihydroxy-16-oxoandrostane and17α-ethynylandrost-5-ene-3β,7β,17β-triol were not appreciably immunesuppressive in this assay at either 300 nM or 30 nM (p>0.05), while17β-aminoandrost-5-ene-3β-ol at 300 nM was moderately immune suppressive(p<0.05) compared to the control reactions. These results show that3β,16α-dihydroxy-17-oxoandrostane, 3β,17β-dihydroxy-16-oxoandrostane and17α-ethynylandrost-5-ene-3β,7β,17β-triol would not be immune suppressivefor lymphocytes in humans in vivo. These results are consistent with thecapacity of the compounds to be anti-inflammatory agents (see, e.g.,example 7) without being immune suppressive.

Example 13

Analysis of immune suppression. Glucocorticoid steroids such asdexamethasone or hydrocortisone are typically immune suppressive andhave significant toxicities associated with their use. Immunesuppression was examined in a reporter antigen popliteal lymph nodeassay in mice essentially as previously described (C. Goebel et al.,Inflamm. Res., 45(Suppl. 2):S85-S90, 1996; R. Pieters et al.,Environmental Health Perspectives 107(Suppl. 5):673-677, 1999). Thisprotocol was used to analyze the activity of17α-ethynylandrost-5-ene-3β,7β,17β-triol in the popliteal lymph node(PLN) assay to show that the compound does not have appreciable immunesuppression activity in vivo. In this protocol, the vehicle was 0.1%carboxymethylcellulose, 0.9% saline, 2% tween 80 and 0.05% phenol, whichcontained 17α-ethynylandrost-5-ene-3β,7β,17β-triol in suspension in drugtreated animals. Assessment of activity included (1) measuringsuppression of numbers of total lymphocytes, antigen specific IgM, IgG1and IgG2a antibody secreting cells (ASC) (ELISPOT assay) in popliteallymph node cells; (2) analysis of cell surface marker (CD4, CD8, CD19,F480, CD80, CD86) expression by flow cytometry of living cells insuspension; and (3) IL-4, TNFα and IFNγ production by lymphocytes invitro (ELISA).

Groups (n=5 per group) of specific pathogen free BALB/C mice were used.The Positive control group was treated with vehicle (oral gavage) and 5μg/day dexamethasone by subcutaneous injection to induce immunesuppression. Vehicle control animals (negative control) were treatedwith vehicle alone (oral gavage). One group of animals was treated with17α-ethynylandrost-5-ene-3β,7β,17β-triol at 0.1 mg/day by oral gavage.Another group was treated with 1 mg/day of17α-ethynylandrost-5-ene-3β,7β,17β-triol was administered to the animalsoral gavage. The results were analyzed by two-tailed Student's t-testwith equal variance. The animals were injected in the right hind footpadwith 50 μL of freshly prepared sensitizing dose of TNP-OVA.Dexamethasone (decadron phosphate injection; dexamethasone sodiumphosphate) was administered by subcutaneous injection into the nape ofthe neck daily, immediately following sensitization with TNP-OVA.17α-Ethynylandrost-5-ene-3β,7β,17β-triol was given immediatelyafterwards by gavage. Five days after injection of TNP-OVA, blood wasdrawn by orbital puncture, and the mice were euthanized by cervicaldislocation and popliteal lymph nodes were removed and separated fromadherent fatty tissue. Single cell suspensions were prepared,resuspended in 1 mL PBS-BSA (1%) and counted. Cell numbers, IL-4, IL-5and IFNγ were measured.

The average number of lymphocytes in PLNs from the vehicle control groupwas 7.8×10⁶ per lymph node compared to 2.9×10⁶ per lymph node in thedexamethasone treated animal group. This reduced lymphocyte countclearly showed the marked immune suppression that is typically seen withthe use of dexamethasone or other glucocorticoid compounds. By contrast,the group treated with 1 mg/day of17α-ethynylandrost-5-ene-3β,7β,17β-triol had 8.2×10⁶ lymphocytes perlymph node and the group treated with 0.1 mg/day of17α-ethynylandrost-5-ene-3β,7β,17β-triol had 11.1×10⁶ lymphocytes perlymph node. The results showed that17α-ethynylandrost-5-ene-3β,7β,17β-triol was not immune suppressive, butwas immune enhancing. 17α-Ethynylandrost-5-ene-3β,7β,17β-triol treatmentat 1.0 mg/day and at 0.1 mg/day increased IFNγ, IL-4 and IL-5 levelscompared to the vehicle control group, also indicating immuneenhancement. The effect of 17α-ethynylandrost-5-ene-3β,7β,17β-triol at0.1 mg/day on IFNγ, IL-4 and IL-5 levels was greater than in the groupthat was treated with 1.0 mg/day. By contrast, IFNγ, IL-4 and IL-5levels were reduced in the dexamethasone treated group compared to thevehicle control group or to either drug treated group.

Example 14

Analysis of immune suppression. Several compounds were characterized fortheir capacity to affect immune responses. This protocol examined theimmune effects of compounds in a standard immune assay. The ovalbumin(OVA) specific immune response assay is a well-established system tomeasure anamnestic (both cell-mediated and antibody-mediated) immuneresponses. BALB/c mice were immunized by intraperitoneal injection(total volume 200 μL) on days 0 and 7 with 100 μg OVA precipitated withalum (25 mg/mL) in saline. Mice (n=5 per group) were treated daily (oralgavage 40 mg/kg, about 1 mg/animal) for 20 days with compound. On day20, blood was drawn and tested in ELISA for antibody titers to OVA. Thecompounds that were tested were 3β,16α-dihydroxy-17-oxoandrostane,16α-bromoepiandrosterone, 17α-ethynylandrost-5-ene-3β,7β,17β-triol,3β,16α-dihydroxyandrostane-17-oxime, 17β-aminoandrost-5-ene-3β-ol and3α,16α,17β-trihydroxyandrostane. None of these compounds were profoundlyimmune suppressive, with OVA antibody titers similar to those in thevehicle control group.

Example 15

Glucose lowering and amelioration of insulin resistance. Glucoselowering effects and amelioration of insulin resistance was assessed inthe diabetic db/db mouse model of human diabetes and insulin resistance.In these studies, db/db C57BL/Ks mice of approximately 8 to 10 weeks ofage were divided into groups of 10 each and then treated with a vehiclecontrol (no drug) or 17α-ethynylandrost-5-ene-3β,7β,17β-triol by oralgavage. The compound was administered twice a day at 20 mg/kg/day (10mg/kg dose administered twice per day), 40 mg/kg/day (20 mg/kg doseadministered twice per day) or 80 mg/kg/day (40 mg/kg dose administeredtwice per day) for up to 28 days. Blood glucose levels were monitoredtwice a week during the dosing period, using a minute amount of blood(nick tail bleeds) to measure the concentration of glucose by glucometerstrips. At specific times during the dosing period (day 14 and day 28),an oral glucose tolerance test (OGTT) was also performed byadministering a standard oral dose of 1 g/kg glucose (approximately 40mg in a 40 mg mouse) and then the fluctuation of blood glucose levelswas monitored quickly thereafter after at 15, 30, 60 and 120 minutesafter the glucose dose. In the drug treated group, an approximately 40%decrease in hyperglycemic blood glucose levels was observed in the db/dbmice. Blood glucose approached 380 mg/dL in the vehicle control groupand was <230 mg/dL after at least 10 days of dosing in the drug treatedgroup. Treatment with drug at 80 mg/kg b.i.d. for 28 days markedlyreduced the peak glycemic excursion from approximately 400 mg/dL 30-minpost-oral glucose dosing seen in vehicle-treated animals down to <200mg/dL in the drug-treated group.

Example 16

Diet induced obesity (DIO) mouse hyperglycemia treatment. The effect ofa drug to enhance peripheral sensitivity to insulin can be studied in amouse model in which a state of insulin resistance is attained byfeeding the animals a fat-enriched diet (60% of total caloric intake)for at least 6 weeks. This model has been described, e.g., J. N. Thupariet al., Proc. Natl. Acad. Sci. USA, 99(14):9498-9502, 2002, H. Xu etal., J. Clin. Invest., 112:1821-1830, 2003, H. Takahashi et al., J.Biol. Chem., 278(47):46654-46660, 2003. Under these diet conditions, themice exhibit increased body weight (+35 g) and a state of glucoseintolerance, which is manifested as a significant delay in the clearancetime of orally-administered glucose during a standard OGTT (oral glucosetolerance test).

For these studies, animals of approximately 4 weeks of age were dividedinto groups of 10 animals each and then treated with a vehicle control(no drug) or 17α-ethynylandrost-5-ene-3β,7β,17β-triol by oral gavage.The 17α-ethynylandrost-5-ene-3β,7β,17β-triol was administered at 20mg/kg, 40 mg/kg or 80 mg/kg twice a day for up to 28 days. At day 14 andday 28 during the dosing period an OGTT was performed. In this DIO-modelof insulin resistance, 17α-ethynylandrost-5-ene-3β,7β,17β-triol notablyreduced glucose intolerance compared to vehicle control animals asindicated by significant improvement in the OGTT glycemic excursion.These findings showed that treatment with17α-ethynylandrost-5-ene-3β,7β,17β-triol enhanced peripheral insulinsensitivity or uptake, which improved glucose intolerance in theseanimals.

Example 17

A treatment protocol similar to that described in example 15 wasperformed with db/db mice that were younger than the animals describedin example 15. The animals (n=8 to 10 per group) were treated with17α-ethynylandrost-5-ene-3β,7β,17β-triol or vehicle by oral gavage twiceper day at 40 mg/kg/day (20 mg/kg dose given twice per day) and 80mg/kg/day (40 mg/kg dose given twice per day). At the start of dosing,the animals were 6 weeks of age, before the onset of elevated glucoselevels or hyperglycemia. Dosing with vehicle or drug was maintained for32 days to determine the effect of the treatments on the onset and rateof progression of hyperglycemia in the animals. In the control group,the onset of hyperglycemia was observed after 25 days of dosing and itcontinued to worsen, i.e., blood glucose levels rose from normal tofrank hyperglycemia, through the end of the 32 day dosing period. Bycontrast, levels of glucose in both drug treatment groups did not riseabove normal levels by the end of the 32 day dosing period, showing thatdrug treatment delayed the onset of hyperglycemia through the course ofthe protocol.

Administration of 17α-ethynylandrost-5-ene-3β,7β,17β-triol to 8 week oldmale diabetic db/db mice markedly suppressed basal blood glucosehyperglycemic levels, an effect that became apparent after 10 days ofdosing and was sustained for 18 additional days of continuous,twice-a-day treatment in the 40 mg/kg dose group. In younger, 6 week oldmale db/db mice, treatment with the17α-ethynylandrost-5-ene-3β,7β,17β-triol at 40 mg/kg completely blockedprogression of the animals into the hyperglycemic state that wasobserved in the vehicle-treated group after 25 days of dosing. Thetreated animals maintained blood glucose levels that were comparable tothose from lean db/+ littermates. Furthermore, results from OGTTsperformed in treated animals model showed significant amelioration ofglucose intolerance compared to vehicle control animals.

Example 18

Glucose lowering in 8 week old db/db diabetic mice. Thehyperinsulinemic-euglycemic clamp protocol was conducted to measureinsulin sensitivity in vivo. In this procedure, insulin was administeredto raise the insulin concentration while glucose was infused to maintaineuglycemia or a fixed, normal blood glucose level (about 180 mg/dL). Theglucose infusion rate (GIR) needed to maintain euglycemia showed insulinaction in these animals. The objective of this protocol was toinvestigate characterize the capacity of17α-ethynylandrost-5-ene-3β,7β,17β-triol andandrost-5-ene-3β,7β,16α,17β-tetrol to ameliorate systemic insulinresistance and improve whole body glucose disposal in thehyperinsulinemic-euglycemic clamp model. The degree of skeletal muscleand hepatic insulin sensitivity and tissue specific glucose uptake werealso assessed. The animals were dosed daily by oral gavage for 14 days.On Day 10 of treatment catheters were implanted in the carotid arteryand jugular vein. On the day of the clamp (day 14) the compound wasadministered at 7:30 am.

Body weight and glucose concentration were assessed on day 0, 7 and day14 of treatment. On day 14 a euglycemic hyperinsulinemic clamp wasperformed. Food was removed at 7:30 am at 10:30 a primed continuousinfusion of [3-³H]-glucose (0.05 μCi/min). A baseline blood sample wastaken at 12:50 (−10 min) and at 1:00 (0 min) a euglycemichyperinsulinemic clamp was initiated by administering 10 mU/kg/min ofinsulin. Glucose was infused at a variable rate to clamp the glucoseconcentration at ˜180 mg/dl. A bolus of [¹⁴C]-2deoxyglucose was given atthe end of the study to assess tissue specific glucose uptake. Plasma¹⁴C2-deoxyglucose was assessed at 122, 125, 130, 135, 145 min. Theanimals were then anesthetized with an intravenous infusion of sodiumpentobarbital and selected tissues were removed, immediately frozen inliquid nitrogen and stored at −70° C. until analysis.

Analysis was conducted as follows. Plasma samples were deproteinizedwith Ba(OH)₂ (0.3 N) and ZnSO₄ (0.3 N), dried and radioactivity wasassessed on scintillation counter (Packard TRICARB 2900 TR, Meriden,Conn.). Frozen tissue samples were homogenized in 0.5% perchloric acid,centrifuged and neutralized. One supernatant was directly counted todetermine radioactivity from both [₁₄C] DG and [¹⁴C] DGP. A secondaliquot was treated with Ba(OH)₂ and ZnSO₄ to remove ¹⁴C DGP and anytracer incorporated into glycogen and then counted to determineradioactivity from free [¹⁴C]DG(2). [¹⁴C]DGP was calculated as thedifference between the two aliquots. The accumulation of [¹⁴C]DGP wasnormalized to tissue weight and tracer bolus. Rg, an index of tissuespecific glucose uptake was calculated as previously described (E. W.Kraegen et al., Am. J. Physiol., 248:E353-E362, 1985). Whole bodyglucose turnover was calculated as the ratio of the ³H glucose infusionrate (dpm/kg/min) and arterial plasma glucose specific activity(dpm/mg). Endogenous glucose production was calculated as the differencebetween the whole body glucose turnover and the exogenous glucoseinfusion rate (R. N. Bergman et al., Endocr. Rev., 6:45-86, 1985).Treatment groups are summarized in the table shown below.

Dosing volume and dosing solution Group Treatment concentration N A -vehicle control* vehicle 8 mL/kg, po, bid for 13 8 mL/kg 10 days, qd onday 14 B - compound 1** 40 mg/kg, po, bid for 13 days, qd 4 mL/kg of 10mg/mL 10 on day 14 stock in vehicle C - compound 1** 80 mg/kg, po, bidfor 13 days, qd 8 ml/kg of 10 mg/ml stock 10 on day 14 in vehicle D -compound 2** 40 mg/kg, po, bid for 13 days, qd 4 mL/kg of 10 mg/mL in 10on day 14 vehicle E - positive*** 25 mg/kg, po, bid for 13 days, qd 5mL/kg of 5 mg/mL in 10 control on day 14 water + 1% CMC *vehicle: 30%sulfobutylether in water (20 mg/mL of drug in solution for groups B-D)**compound 1: 17α-ethynylandrost-5-ene-3β,7β,17β-triol compound 2:androst-5-ene-3β,7β,16α,17β-tetrol ***rosiglitazone maleate (31493r,AApin Chemicals Limited (UK), CMC—Carboxymethyl cellulose (medium grade,C4888, Sigma)

The insulin dose was 10 mU/kg/min. In a normal animal, this dose ofinsulin would require infusion of ˜90 mg/kg/min of glucose to keep theglucose level clamped at ˜150 mg/dl. The average glucose requirement inall treatment groups was ˜50% of normal. The results showed that both17α-ethynylandrost-5-ene-3β,7β,17β-triol andandrost-5-ene-3β,7β,16α,17β-tetrol increased the glucose infusion ratecompared to the vehicle control, which means insulin action was improvedin the groups B, C, D and E.

Using the 3-³H glucose tracer, the rate of liver glucose production wascalculated during the basal period and the ability of insulin tosuppress liver glucose production during the clamp. In severe insulinresistant animals endogenous glucose production would decrease by about50% with the insulin dose that was used. In groups C, D and E, insulincompletely suppressed endogenous glucose production (p<0.05), whichshowed an improvement in hepatic insulin action.

To assess peripheral insulin action, tissue specific glucose uptakeduring the euglycemic hyperinsulinemic clamp was assessed using¹⁴C-2-deoxyglucose. A bolus of ¹⁴C-2-deoxyglucose was given at 120 min.Tissues were collected 25 minutes later. Tissues were analyzed for totalaccumulation of ¹⁴C-2-deoxyglucose phosphate. In this protocol, brainglucose uptake is unaffected by most treatment regimens and it thusserves as an internal control. The results showed that brain glucoseuptake was comparable between all of the groups. In the heart anddiaphragm, glucose uptake was higher in the treated groups compared tothe vehicle control group. Both androst-5-ene-3β,7β,16α,17β-tetrol androsiglitazone were more effective (p<0.05) in augmenting muscle glucoseuptake in the gastrocnemius muscle. In white vastus muscle, which is anon oxidative muscle group, differences were not detected except betweenandrost-5-ene-3β,7β,16α,17β-tetrol and rosiglitazone.

Example 19

Rats were fed ad libum with a standard laboratory chow that contained0.45% wt/wt of androst-5-ene-3β,7β,17β-triol for 6 days, followed byanalysis of liver tissue on day 6 for levels of phosphoenolpyruvatecarboxykinase (“PEPCK”) and 11β-hydroxysteroid dehydrogenase (“11β-HSD”)in the liver. Control animals were fed normal chow and livers wereexamined on day 6 for PEPCK and 11β-HSD levels by measurement ofmessenger RNAs (mRNAs) by RT-PCR. Both control and treated animals hadfree access to water. Administration of the compound in chow for 6 dayswas found to decrease levels of 11β-HSD type 1 (“11β-HSD1”) and PEPCK inliver tissue as shown below. Levels of PPARα mRNA in these animals werenot affected by feeding with androst-5-ene-3β,7β, 17β-triol.

11β-HSD1 PEPCK PPARα mRNA mRNA mRNA control (no compound) 100% 100% 100%androst-5-ene-3β,7β,17β-triol  45%  30% 105%

In another study, administration of the compound17α-ethynylandrost-5-ene-3β,7β,17β-triol to mice was found to decreaseexpression of 11β-HSD1 in osteoblasts by about 50%, which is consistentwith the observation that the compound possesses bone-sparing effects inmice treated with dexamethasone, a glucocorticoid that induces bone lossin vivo.

In another study, total RNA from perigonadal fat tissue from lean db/+or diabetic db/db mice treated with 20 mg/kg of17α-ethynylandrost-5-ene-3β,7β,17β-triol was isolated and processed forquantitative RT/PCR using primers specific for monocyte chemoattractantprotein-1 (MCP-1) using an iCycler iQ multicolor real time-detectionsystem (Bio-Rad). RNA expression levels were normalized with respect tothe vehicle control. The compound was found to decrease levels ofmonocyte chemoattractant protein-1 (MCP-1) by about 50%. For this study,vehicle was also administered to a control group of age matched leanheterozygous db/+ mice (n=7).

Other compounds such as 17α-ethynylandrost-5-ene-3β,7β,17β-triol,androst-5-ene-3β,7β,16α,17β-tetrol, androst-5-ene-3β,7α,16α,17β-tetrol,androst-5-ene-3α,7β,16α,17β-tetrol, androst-5-ene-3β,4β,16α,17β-tetrol,androst-5-ene-3α, 4β,16α,17β-tetrol or monoesters or diesters of thesecompounds, e.g., compounds containing one or two acetate or propionateesters at the 3- or 17-positions, are examined in a similar manner fortheir capacity to decrease the level or activity of PEPCK or a 11β-HSD,such as 11β-HSD type 1 or 11β-HSD type 2, in hepatocytes orliver-derived cells or in other tissues or cells such as kidney, muscle,bone tissue or cells, adipose tissue or cells or CNS tissue or cells,e.g., neurons or glia.

Example 20

Inhibition of the generation of CD4⁺CD25⁺ T regulatory cells or theiractivity in vivo. Purified CD4⁺CD25⁻ T cells (5×10⁶ cells) from congenicB6.5JL mice (CD45.1) per group were adoptively transferred into each offive B6 mice (CD45.2). The purified CD4⁺CD25⁻ T cells were obtained byfluorescence activated cell sorting (FACS) of the donor cells at leasttwice. Vehicle (0.1% carboxymethylcellulose, 0.9% saline, 2% tween 80,0.05% phenol) or 16α-bromoepiandrosterone in vehicle (1 mg/animal/day in100 μL vehicle) was injected subcutaneously before transfer of the cellsfrom the CD45.1 donor animals and the injections were continued dailyfor 14 days. Thymus, lymph nodes and spleens were collected from theanimals at day 15. Samples of thymus, lymph node and spleen wereobtained from individuals, and the cells were labeled with fluorescentantibody that bound to CD4, CD25, CD103 or Foxp3. The cells were thenanalyzed by flow cytometry to enumerate the numbers of the various celltypes. The remainder of cells from lymph nodes and spleen of eachtreatment group were pooled, pre-enriched for CD4⁺CD25⁺ cells and thenanalyzed for CD4⁺CD25⁺ that arose from the host (endogenous CD45.2cells) and from donor cells (CD45.1 cells that converted from theCD4⁺CD25⁻ donor phenotype to the CD4⁺CD25⁺ phenotype after residing invivo for 15 days). The cells were analyzed by cell sorter. To test forregulatory function, varying numbers of purified converted CD45.1 orendogenous CD45.2 CD4⁺CD25⁺ cells were co-cultured with 2000 CD4⁺CD25⁻responder cells, 1×10⁵ irradiated spleen cells as antigen presentingcells, and 0.5 mg/ml of anti-CD3 antibody. Fresh CD4⁺CD25⁺ cells wereused as controls. Proliferation was determined by measurement of³H-thymidine uptake 4 days after initiation of culture.

The results showed that the number of donor CD45.1 CD4⁺CD25⁺ Treg cellsin the spleens from drug treated animals was lower than the number ofCD45.1 CD4⁺CD25⁺ Treg cells in the spleens from vehicle control animals.The average vehicle control CD45.1 CD4⁺CD25⁺ cell number was 1.97×10⁵cells compared to an average of 0.62×10⁵ CD45.1 CD4⁺CD25⁺ cells from thedrug treated animals.

The number of endogenous CD45.2 CD4⁺CD25⁺ Treg cells in the spleens fromdrug treated animals was also lower than the number of CD45.1 CD4⁺CD25⁺Treg cells in the spleens from vehicle control animals. The averagevehicle control CD45.2 CD4⁺CD25⁺ cell number was 9.54×10⁶ cells comparedto an average drug treated 5.49×10⁶ CD45.2 CD4⁺CD25⁺ cells.

The average endogenous CD45.2 CD4⁺CD25⁺ cells in the thymus of vehiclecontrol animals was 3.10×10⁵ compared to 1.59×10⁵ in the drug treatedanimals.

The percent of donor CD45.1 CD4⁺CD25⁺CD103⁺ cells compared to totaldonor CD4⁺CD25⁺ cells in the spleens from drug treated animals was lowerthan the number of CD45.1 CD4⁺CD25⁺CD103⁺ Treg cells compared to totaldonor CD4⁺CD25⁺ cells in the spleens from vehicle control animals. Theproportion of endogenous CD45.2 CD4⁺CD25⁺CD103⁺ Treg cells was about thesame in spleens from vehicle control animals (13.85%) compared to drugtreated animals (13.40%). The average of donor CD45.1 CD4⁺CD25⁺CD103⁺cells for vehicle controls was 29.06% compared to an average of 9.63% inthe drug treated animals. The CD103 surface antigen is expressed byactivated Treg cells. This indicated that the relative proportion ofactivated CD45.1 CD4⁺CD25⁺ cells was lower in the drug treated animalsthan in the vehicle controls, which is consistent with inhibition ofTreg cell activity for the donor cells in vivo.

Suitable variations of this protocol include (1) the use of a highernumber of donor CD4⁺CD25⁻ T cells per animal, e.g., 1×10⁶/animal,1.5×10⁶/animal or 2×10⁶/animal, (2) different daily dosages of the drug,(3) a different route of administration of the drug, (4) a differentcompound as the drug and (5) inclusion of additional groups of animals,e.g., a group that receives another therapeutic agent such as anantiinflammatory or immune suppressive glucocorticoid such asdexamethasone or cortisol. Some of these variations can apply to theprotocols at example 3 or the some of cited references.

Example 21

Increase of CD4⁺CD25⁺ T regulatory cells or their activity in vivo. Thecompound 17α-ethynylandrost-5-ene-3β,7β,17β-triol was administered tomice essentially as described in a previously described collagen inducedarthritis animal model. H. Offner et al., Clin. Immunol., 110:181-190,2006.

DBA/1 Lac/J mice were used for the study. The mice were obtained fromJackson Laboratories (Bar Harbor, Harbor, Mass.) and housed inaccordance with applicable institutional guidelines. Bovine type IIcollagen (bCII) was used to induce collagen induced arthritis (CIA) byimmunizing 8-week-old mice with 200 μg of bCII emulsified 1:1 with CFAcontaining 200 μg Mycobacterium tuberculosis (100 μL; Difco, Detroit,Mich.). The antigen was injected intradermally at the base of the tail.The animals were monitored for 4-7 weeks to observe the onset andprogression of the disease post-immunization. The arthritic severity wasevaluated with a grading system for each paw according to the followingscale: 0=no redness or swelling; 1=slight swelling in ankle or rednessin foot; 2=progressed swelling and inflammation and redness from ankleto mid foot; 3=swelling and inflammation of entire foot; 4=swelling andinflammation of entire foot including toes.

After immunization, the mice were treated with the drug at 40 mg/kg/dayin vehicle by oral gavage beginning at the start of observable clinicaldisease (beginning at about 26-27 days after immunization). The vehiclethat was used for the protocol was 30% cyclodextrin-sulfobutylether inwater. The cyclodextrin-sulfobutylether was obtained commercially(Captisol™ available at cyclexinc.com). The drug formulation was 20 mgdrug/mL in the vehicle.

The results obtained from drug treated animals indicated thatadministration of the 17α-ethynylandrost-5-ene-3β,7β,17β-triol increasedthe frequency of Foxp3⁺ and CD4⁺Foxp3⁺ expressing cells in wholesplenocytes as shown below.

Vehicle (n = 3) drug (n = 3) p value Total Foxp3⁺ 1.6% ± 0.16 2.39% ±0.16 0.00001 CD4⁺Foxp3⁺ 1.1% ± 0.09 1.34% ± 0.05 <0.00001

Cell sorter analysis showed an increase in Foxp3+ CD4⁺ cells in drugtreated animals (1.4%) relative to control animals (1.0%). The Foxp3protein is associated with differentiation or conversion of CD4⁺CD25⁻ Tcells to CD4⁺CD25⁺ Treg cells and an increase in the number of cellsexpressing Foxp3 indicates increased development of Treg cells fromtheir precursor cells. After immunization, the mice were treated withthe drug at 40 mg/kg/day in vehicle by oral gavage beginning at thestart of observable clinical disease (beginning at about 26-27 daysafter immunization). The results obtained from drug treated animalsindicated that administration of the17α-ethynylandrost-5-ene-3β,7β,17β-triol increased the frequency ofFoxp3⁺ and CD4⁺Foxp3⁺ expressing cells in whole splenocytes as shownbelow. Consistent with this was a statistically improved clinical scorein the drug treated animals compared to vehicle controls at days 44-49after immunization. Between days 34-49 the vehicle control animals had amean clinical score of about 6.8-8 while the drug treated animals had amaximum mean clinical score of about 5 at day 34 with a slow decline toa mean score of about 3 by day 49. These results indicated that thecompound slowed the progression of arthritis and reduced its maximumseverity compared to vehicle control animals.

Example 22

Synthesis of compounds is described below.

Androst-5-ene-3β,7β,16α,17β-tetrol (7)

5-androstene-3β,16α-diol-17-one diacetate (3).16α-bromodehydroepiandrosterone 2 was prepared by refluxing DHEA (1) inmethanol with copper (II) bromide. To 15.0 g of 2 (40.8 mmol) inpyridine (129 mL) and water (309 mL) was added 120 mL of 1N aqueoussodium hydroxide and the mixture was stirred in air for 15 minutes. Thereaction mixture was poured into ice/water saturated with sodiumchloride and containing excess hydrochloric acid. The crude product wasfiltered, washed with water until neutral and dried in vacuo overanhydrous calcium chloride at 55-60° C. Recrystallization from methanolafforded 8.21 g of 16α-hydroxy-DHEA (Mp 194.4-195.1° C.). This productwas then converted to the diacetate 3 by treatment with excess aceticacid in pyridine and purified by flash chromatography.

5-Androstene-3β,16α-diol-7,17-dione (5). To a solution of 3 (20.1 g,51.7 mmol) in benzene containing celite (60 g) and pyridinium dichromate(75 g) was added 22 mL of 70% tert-butyl hydrogen peroxide. After 2 daysof stirring at room temperature, diethyl ether (600 mL) was added andprecipitate was filtered and washed with ether (2×100 mL). The residuewas purified by flash chromatography (60% ethyl acetate in hexanes) andrecrystallized to give 16.0 g (39.8 mmol, 77%) of 5 as prisms. Mp205.6-206.2° C.

5-Androstene-3β,7β,16α,17β-tetrol (7). To a solution of 5 (10.0 g, 24.8mmol) in dichloromethane (75 mL) and methanol (255 mL) at 0° C. wasadded 1.5 g of sodium borohydride and the mixture was stirred at 0° C.for 1 hour. After quenching with acetic acid (3.5 mL) the reactionmixture was partitioned between dichloromethane and water. The organiclayer was concentrated to a mixture of 7α and 7β diacetate tetrols. Thismixture was purified by flash chromatography and HPLC to give 2.90 g ofthe 7β-epimer (9.5 mmol, 38%). Mp 216.8-220.8° C. Saponification inmethanol (100 mL) with 1N sodium hydroxide (60 mL) for 2 days at roomtemperature and purification by HPLC gave 7 (1.41 g, 4.4 mmol, 46%) asfine needles from aqueous acetonitrile. Mp 202.1-206.4° C.; [a]D+1.35(methanol, c=1). Selected ¹H NMR peaks (CD₃OD): d 0.77 (s, 3H), 1.01 (s,3H), 3.39 (d, 1H), 3.46 (m, 1H), 3.74 (t, 1H), 4.04 (m, 1H), 5.55 (dd,1H).

3α,7α,17β-Triacetoxyandrost-5-ene-16α-ol (8),androst-5-ene-3α,7α,16α,17β-tetrol (9)

16α-Bromo-5-androstene-3α-ol-17-one (2). A solution of5-dehydroandrosterone (1) (17.8 g, 61.7 mmol) in methanol (1.35 L) wasrefluxed with copper (II) bromide (36.4 g, 163 mmol) with stirring for19 hours. To the cooled reaction mixture was added water (1.35 L) anddichloromethane (1.5 L). The organic layer was filtered throughanhydrous sodium sulfate and the product crystallized as fine needlesfrom methanol (16.7 g, 45.5 mmol, 74%). Mp 195-207° C.

3α,16α-Diacetoxy-5-androsten-17-one (4). To a solution of 2 (12.0 g,32.7 mmol) in pyridine (1.032 L) and water (0.247 L) in air was addedaqueous 1N sodium hydroxide (90 mL) and the mixture was stirred at roomtemperature for 15 minutes. The reaction mixture was added to anice/water mixture containing 1.2 L of 1N hydrochloric acid. Aftersaturating the solution with sodium chloride, it was extracted withethyl acetate (2×1 L). The combined organic layers were washed withbrine (250 mL), filtered through anhydrous sodium sulfate andconcentrated. The crude 5-androstene-3α,16α-diol-17-one (3) was treatedwith excess acetic anhydride in pyridine at room temperature overnightand purified by column to give 4 (7.46 g, 19.2 mmol, 59%) as prisms frommethanol. Mp 172.7-173.7° C.

5-Androstene-3α,16α,17β-triol 3,16-diacetate (5). To a solution ofenediolone diacetate 4 (7.46 g, 19.2 mmol) in dichloromethane (45 mL)and methanol (120 mL) at 0° C. was added sodium borohydride (950 mg).The solution was stirred at 0° C. for 1 hour. After addition of excessacetic acid the reaction mixture was partitioned between dichloromethaneand water. The organic layer was filtered through anhydrous sodiumsulfate and concentrated to yield a mixture of the 17α (minor) and 17β(major) epimers. This mixture was purified by flash chromatography (25%ethyl acetate in hexanes) to give 6.1 g (15.6 mmol, 81%) of the 17βepimer 5. Mp 126.9-128.6° C. The triacetate 6 was made from 5 treatedwith excess acetic anhydride in pyridine at room temperature overnightand was purified by column to give 6.0 g (13.9 mmol, 89%).

5-Androstene-3α,16α,17β-triol-7-one triacetate (7). A solution of thetriacetate 6 (6.0 g, 13.9 mmol) in benzene (255 mL) was treated withcelite (25.5 g), pyridinium dichromate (31.5 g) and 70% tert-butylhydrogen peroxide (9.0 mL) and stirred at room temperature for 19 hours.Anhydrous diethyl ether (255 mL) was added and reaction mixture wascooled in an ice bath for 1 hour. The resulting solid was filtered offand washed with ether (2×50 mL). The combined organic portions wereconcentrated and purified by flash chromatography (29% ethyl acetate inhexanes) to give 3.45 g of 7 (7.7 mmol, 55%).

5-Androstene-3α, 7α,16α,17β-tetrol (9). To a solution of 7 (3.45 g, 7.7mmol) in dichloromethane (15 mL) and methanol (30 mL) at 0° C. was addedsodium borohydride (1.0 g) and the solution was stirred at 0° C. for 2hours. After addition of excess acetic acid (1.5 mL) the reactionmixture was partitioned between dichloromethane and water. The organiclayer was filtered through anhydrous sodium sulfate and concentrated toyield a mixture of the 7α (minor) and 7β (major) epimers. This mixturewas saponified in methanol (100 mL) with 1N sodium hydroxide (60 mL)overnight at room temperature. The crude tetrols were recovered bypartitioning the saponification mixture between ethyl acetate and brine.The epimers were separated by HPLC to give 220 mg of 9 (0.68 mmol, 9%).Mp 243-248.3° C.). Selected ¹H NMR peaks (CD₃OD): δ 0.77 (s, 3H), 1.02(s, 3H), 2.11 (m, 1H), 2.57 (m, 1H), 3.34 (s, 1H), 3.44 (d, 1H), 3.70(brt, 1H), 4.04 (m, 2H), 5.55 (dd, 1H). The epimers of 8 are separatedby HPLC to obtain purified 8 and its 7β-acetete epimer.

Androst-5-ene-3β,7β,11β,17β-tetrol-3β-acetate (8),androst-5-ene-3β,7β,11β,17β-tetrol (9),androst-5-ene-3β,7β,17β-tetrol-3β-acetate-11-oxime (10)

I: To a solution of 1 (4 g) in 150 ml Ac₂O, was added p-TsOH 2.8 g, atroom temperature, overnight, work up with adding 700 mL ice water,stirring for 1 hr until solid formed, filtered out to yield white solidproduct 2, 4.55 g

II: To a solution of 1.5 g NaBH4 in 35 ml EtOH and 5 ml MeOH, was slowlyadded a solution of 2 (1.2 g) in 30 ml EtOH and 10 ml chloroform at 0°C. The solution was continued with stirring for 2 hrs at 0° C., then atroom temperature 2 hrs. After this time 4 ml acetic acid was added toquench NaBH4, then 50 ml water. The product was isolated by extractingwith EtoAc 50 ml×3, removal of solvent in vacuo to yield crude product.Purification was accomplished via column chromatography to yield 3, 250mg.

III: To a solution of 3 (200 mg) in 8 ml MeOH, was added a solution of0.36 g H5IO6 in 2 ml water, stirring for 1 hr at room temperature,removal of solvent in vacuo, addition of water and dicholormethaneextraction. Purification used column chromatography to yield product 4,60 mg.

IV: To a solution of 4 (0.4 g) in 5 ml pyridine was added 0.5 mlacetetyl chloride, slowly at 0° C., stirring continued for 15 min at 0°C., then room temperature for 30 min. The reaction was quenched byadding 20 ml water, extraction with EtoAc 15 ml×3, washing with 1N HCl,saturated NaHCO3, brine, then dries over Na2SO4. Concentration in vacuogave a yield of 5, 520 mg.

V: To a solution of 5 (0.5 g) and 0.13 g CuI in 15 ml acetonitrile, wasadded 3 ml 70% t-BuOOH slowly, stirring for 1 hr then 50° C. for 2 hrs.Add 12 ml 10% Na2S2O5 solution, extract with EtOAc, dry over Na2SO4,remove solvent, run column to yield 6, 80 mg.

VI: To a solution of 6 (50 mg) in 1.5 ml THF and 3 ml MeOH, was added260 mg CeCl3.7H20, then added 75 mg NaBH4 slowly at 0° C., stirring for30 min, add 0.5 mL 1N HCl and 5 ml water, extract with EtoAc 5 ml×3, dryover Na2SO4, remove solvent to yield 7, 49 mg.

VII: To a solution of 300 mg NaBH4 in 4 ml EtOH and 1 ml MeOH, was addeda solution of 7 (40 mg) in 0.5 ml EtOH and 0.5 ml chloroform, stirringfor 8 hrs at 0° C., then in a freezer overnight. Add acetic acid toquench reaction, add water and EtoAc extraction to yield 8, 30 mg.mp>250° C.; ¹H NMR (CD₃OD) δ 0.86 (s, 3H), 1.35 (s, 3H), 1.95 (s, 3H),3.55 (t, 1H, J=7.5 Hz), 3.71 (dd, 1H, J=7 Hz, J=2.5 Hz), 4.32 (d, 1H,J=2.7 Hz), 4.55 (m, 1H), 5.21 (s, 1H)

VIII: To a solution of 8 (30 mg) in 1 mL MeOH, was added a solution of50 mg NaOH in 0.25 mL water. Stirring for 15 min at 50° C., then add 1NHCl 1 mL, water 5 mL, EtoAc 5 mL×3 to extract, remove solvent to yield9, 20 mg. mp 170-172° C.; ¹H NMR (CD₃OD) δ 0.95 (s, 3H), 1.32 (s, 3H),3.41 (m, 1H), 3.51 (t, 1H, J=8.0 Hz), 3.78 (dd, 1H, J=7.1 Hz, J=2.5 Hz),4.31 (d, 1H, J=2.5 Hz), 5.15 (s, 1H)

IX: To a solution of 29 mg NH₂OH.HCl and 17 mg NaOH in hot 1 mL EtOH,was added a solution of 9 (50 mg) in hot 1 mL EtOH, refluxing for 2 hrsat 100° C., filtered out salt, recrystallized in EtOH/H₂0 to yield 10,40 mg. mp>250° C.; ¹H NMR (CD₃OD) δ 0.72 (s, 3H), 1.02 (s, 3H), 2.03 (s,3H), 3.86 (t, 1H, J=8.5 Hz), 4.08 (dd, 1H, J=8.0 Hz, J=2.6 Hz), 4.60 (m,1H), 5.19 (s, 1H)

17α-Methylandrost-5-ene-3β,17β-diol-3β-acetate-7,11-dione (7),17α-methylandrost-5-ene-3β,7β,17β-triol-3β-acetate-11-one (8),methylandrost-5-ene-3β,7β,17β-triol-11-one (9)

I: To a solution of 1 (4 g) in 150 ml Ac2O, was added p-TsOH 2.8 g, RT,o/n, work up with adding 700 mL ice water, stirring for 1 hr, filteredsolid out to yield a white product 2, 4.55 g.

II: To a solution of 1.5 g NaBH₄ in 35 mL EtOH and 5 mL MeOH, was addeda solution of 2 (1.2 g) in 30 mL EtOH and 10 mL chloroform at 0° C.,slowly, continued to stir for 2 hrs at 0° C., and RT 2 hrs, added 4 mLacetic acid to quench NaBH₄, add 50 mL water, extracted with EtoAc 50mL×3, then removed solvent to yield crude product. Column yield 3, 250mg.

III: To a solution of 3 (200 mg) in 8 ml MeOH, was added a solution of0.36 g H5IO6 in 2 ml water, stirred for 1 hr at RT, removed solvent, addwater and DCM extraction, then ran column to yield product 4, 60 mg.

IV: To a solution of 4 (250 mg) in 1.5 mL THF and 3.5 mL ether at −78°C. under N₂, was added 1 mL 22% MeMgCl in THF slowly, stirring for 1.5hrs at −78° C., then RT for 1 hr, then refluxed for 1 hr at 75° C. Added4 mL 1N HCl and 10 mL water at 0° C. EtoAc extraction, removed solventto yield crude 249 mg. Column run yielded 5, 46 mg.

V: To a solution of 5 (1.0 g) in 15 mL pyridine was added 1.1 mL aceticchloride slowly at 0° C., stirring for 15 min at 0° C., then RT for 30min. add 50 mL water, extracted with EtoAc 50 mL×3, washed with 1N HCl,Sat NaHCO3, brine and dried over Na₂SO₄. Solvent removal yielded 6, 1.02g.

VI: To a solution of 6 (1.0 g) and 0.3 g CuI in 40 mL acetonitrile, wasadded 6 mL 70% t-BuOOH slowly, stirring for 1 hr at RT then at 50° C.for 2 hrs. Added 24 mL 10% Na₂S₂O₅ solution, extracted with EtoAc, dryover Na₂SO₄, removed solvent, ran column to yield 7, 285 mg. mp>250° C.;¹H NMR (CD₃Cl) δ 0.82 (s, 3H), 1.29 (s, 3H), 2.05 (s, 3H), 4.70 (m, 1H),5.75 (s, 1H)

VII: To a solution of 7 (45 mg) in 1.5 mL THF and 3 mL MeOH, was added150 mg CeCl₃.7H₂0, then added 30 mg NaBH₄ slowly at 0° C., stirring for10 min, add 0.5 mL 1N HCl and 5 mL water, extracted with EtoAc 5 mL×3,dried over Na₂SO₄, removed solvent to yield 8, 41 mg. mp 108-110° C.; ¹HNMR (CD₃OD) δ 0.725 (s, 3H), 1.25 (s, 3H), 2.01 (s, 3H), 4.02 (dd, 1H,J=8.2 Hz, J=2.4 Hz), 4.53 (m, 1H), 5.29 (s, 1H)

VIII: To a solution of 8 (22 mg) in 1 mL MeOH, was added a solution of23 mg NaOH in 0.1 mL water. Stirred for 10 min at 50° C., then added 1NHCl 1 mL, water 5 mL, EtoAc 5 mL×3 to extract. Removed solvent to yield9, 10 mg. mp>250° C.; ¹H NMR (CD3OD) δ 0.75 (s, 3H), 1.24 (s, 3H), 3.41(m, 1H), 3.99 (dd, 1H, J=8.2 Hz, J=2.5 Hz), 5.23 (s, 1H)

17α-Ethynylandrost-5-ene-3β,7β,16α,17β-tetrol (8),17β-ethynylandrost-5-ene-3β,7β,16α,17α-tetrol (9)

I: To a solution of 1 (1.0 g) and 0.56 g imidazole in 15 ml DMF, wasadded TBDMS-Cl 1.24 g, RT, o/n, work up with adding 50 ml water, solidshow up, filtered out to yield white solid product 2, 1.75 g.

II: To a solution of 2 (1.64 g) in 50 ml THF cooled to −78° C., wasadded 2.3 ml LDA, 30 min later, was added 0.62 ml TMSCl slowly, stirringfor 30 min at −78° C., then warm up to RT stirring for 1 hr, TLC showsRXN was completed, extraction with ether 150 ml×2, washed with water andbrine, dried over Na2SO4 to yield yellow product 3, 1.87 g.

III & IV: To a solution of 3 (10 g) in 250 ml THF cooled to −20° C., wasadded m-CPBA 4.2 g, stirring for 3 hrs to form 4, then add 250 ml MeOHslowly, stirring for 30 min at −20° C., then adding 200 ml Na2SO3solution slowly at −20° C., stirring for 1 hr. Warm up to RT, extractwith ether 150 ml×3, washed with Sat NaHCO3, brine, dry over Na2SO4 toyield crude 11 g, in order to remove some extra m-CPBA in the product,run short column, 100% Hex 50 ml×5, then 50% Hex/EtoAc 100 ml×5 tocollect crude product 5, 8.5 g.

V: To a solution of 10 g 90% lithium acetylide ethylene diamine complexin 250 ml dry THF, was added a solution of 5 (5 g) in 50 mL dry THF bysyringe pump, which took about 8 hrs. Let it stir for o/n at RT. Added500 mL water at 0° C., extracted with EtoAc 150 mL×3, washed with 200 mL0.1N HCl, 150 mL saturated NaHCO₃, 100 mL brine, dry over Na₂SO₄, removesolvent to yield crude 5.5 g, run column to collect two isomers,17-β-OH, 6 (1.5 g) and 17-α-OH, 7 (1.2 g).

VI: To a solution of 6 (265 mg) in 4 mL MeOH and 3 mL THF, was added 4mL 1N HCl at RT, 1.5 hrs. Added 10 mL saturated NaHCO₃, removed organicsolvent at RT, added 10 mL water, stored in freezer o/n to remove water,added THF to the solid, filtered, removed THF to yield a white solidproduct 8, 130 mg. mp 214-216° C.; ¹H NMR (CD₃OD) δ 0.92 (s, 3H), 1.06(s, 3H), 2.99 (s, 1H), 3.42 (m, 1H), 3.72 (dt, 1H, J=7.2 Hz, J=2.5 Hz),4.17 (dd, 1H, J=8.2 Hz, J=2.7 Hz), 5.24 (d, 1H, J=1.0 Hz).

VII: To a solution of 7 (500 mg) in 8 mL MeOH and 6 mL THF, was added 8mL 1N HCl at RT for 1.5 hrs. Added saturated NaHCO₃ to neutralize thesolution pH=8. Added 50 ml water to obtain a white solid, filtered,washed with water, dried over vacuum to yield the white solid product 9,225 mg. mp>250° C.; ¹H NMR (CD₃OD) δ 0.90 (s, 3H), 1.06 (s, 3H), 2.75(s, 1H), 3.42 (m, 1H), 3.72 (dt, 1H, J=7.0 Hz, J=2.0 Hz), 4.37 (dd, 1H,J=8.1 Hz, J=2.6 Hz), 5.24 (t, 1H, J=2.0, J=1.0 Hz).

4β-Acetylandrost-5-ene-3β,16α,17β-triol (7),androst-5-ene-3β,4β,16α,17β-tetrol (compound 8)

Step 1: A mixture of compound 1 (24.0 g, 0.0832 mol) and copper bromide(56.0 g, 0.20 mol) in anhydrous methanol (800 mL) was refluxed for 16hr. Most of solvent was removed under vacuum and water (500 mL) wasadded. The resulting precipitate was collected by filtration and washedwith water. The solid was recrystallized in methanol twice to affordcompound 2 as a pale yellow solid (19.7 g)

Step 2: To a stirring solution of compound 2 (22.0 g, 0.060 mol) in 200mL N,N-dimethylformamide was added 1 N sodium hydroxide aqueous solution(66 mL, 0.066 mol). The reaction mixture was stirred at room temperaturefor 1 hr. 1N aqueous hydrochloric acid (8 mL) and 400 mL water wereadded. The resulting precipitate was collected by filtration and washedwith water. Purification of this crude product by recrystallization frommethanol to afford compound 3 as a white solid (11.8 g).

Step 3: To a solution of compound 3 (11.8 g, 0.0387 mol) in pyridine (50mL) was added acetyl chloride (11.8 g, 0.128 mol) at 0° C. The reactionmixture was stirred at 0° C. for 1 hr. The resulting mixture was warmedup to room temperature and stirred for another 1 hr. Water was added.The precipitate was collected by filtration and washed with water. Thesolid was dried over vacuum to give compound 4 (12.6 g) which wascarried on without further purification.

Step 4: Lithium aluminum hydride (1.13 g, 0.030 mol) was added to a cold(0° C.) solution of compound 2 (3.10 g, 0.00916 mol) in 80 mL ofanhydrous ether under nitrogen. The ice bath was removed and theresulting mixture was stirred at room temperature for 0.5 h and thenrefluxed for 1 h. The reaction was quenched by the addition of 6 Naqueous hydrochloric acid. Ether was removed under reduced pressure. Theresulting solid was filtered and washed with water. The crude productwas recrystallized in methanol to afford compound 5 (1.1 g) as a whitesolid.

Step 5: To the solution of 5 (914 mg, 2.98 mmol) in 20 mL of chloroformwas added bromine (303 mg, 3.16 mmol). The reaction mixture was stirredat room temperature for 20 min. Solvent was removed in reduced pressureto give compound 6 which was carried on without further purification.

Step 6: Preparation of 4β-acetylandrost-5-ene-3β,16α,17β-triol (7).Compound 6 was dissolved in 30 mL of anhydrous ether and 10 mL ofanhydrous pyridine. A solution of silver acetate (1.03 g, 1(914 mg, 2.98mmol) in 5 mL of anhydrous pyridine was added. The reaction mixture wasstirred under dark for 0.5 hr. A heavy greenish precipitate wasdeposited. Ether (50 mL) was added and precipitate was filtered off. Thefiltrate was under vacuum to dryness. The residue was purified by flashchromatograph on silica gel eluted with 50:50 ethyl acetate:hexanes toafford the title compound 7 (124 mg) as a white solid. Selected ¹H NMRdata: (CD₃OD, 300 MHz): δ 5.78 (d, 1H, J=2.2 Hz), 5.34 (br, 1H), 4.02(t, 1H, J=4.5 Hz), 3.52 (dt, 1H, J=7.7 Hz, 3.0 Hz), 3.37 (d, 1H, J=4.9Hz), 2.03 (s, 3H), (1.12 (s, 3H), 0.75 (s, 3H). Melting Point: 152-153°C.

Step 7: Preparation of androst-5-ene-3β,4β,16α,17β-tetrol (8). Thecompound 7 (50 mg, 0.137 mmol) was dissolved in 1 N sodium hydroxideaqueous (1 mL) and methanol (5 mL) and the resulting solution wasrefluxed for 1 hr. Methanol was removed under vacuum and the residue wasextract with ethyl acetate (3×30 mL). The combined extracts were driedover magnesium sulfate, filtered, and concentrated under vacuum toafford a solid. The crude product was purified by recrystallization frommethanol to afford title compound 8 ((23 mg) as a white solid. Selected¹H NMR data: (CD₃OD, 300 MHz): δ 5.62 (d, 1H, J=3.2 Hz), 4.05 (d, 1H,J=2.4 Hz), 4.02 (m, 1H), 3.43 (dt, br, 1H, J=11.7 Hz, 3.6 Hz), 3.36 (d,1H, J=4.2 Hz), 1.197 (s, 3H), 0.76 (s, 3H). Melting Point: 238-241° C.

Androst-5-ene-3β,4β,7β,17β-tetrol (12) (method 2),androst-5-ene-3β,3β,7β,17β-triacetoxy-4β-ol (11)

Step 1: To a solution of compound 9 (5.0 g, 0.0138 mol) in pyridine (20mL) was added acetyl chloride (11.8 g, 0.128 mol) at 0° C. The reactionmixture was stirred at 0° C. for 5 hr then most solvent was removedunder vacuum. The residual sludge was partitioned between ethyl acetate(80 ml) and water (20 ml). The organic layer was washed with 1N aqueoushydrochloric acid, saturated sodium bicarbonate aqueous solution thendried over magnesium sulfate, filtered, and evaporated to a solid. Thecrude product was recrystallized from ethyl acetate and hexane to affordcompound 10 (4.8 g) as white solid.

Step 2: To a solution of compound 10 (720 mg, 1.66 mmol) in dioxane (15mL) and acetic acid (10 mL) was added selenium dioxide (185 mg, 1.66mmol) in water (1.5 mL) and dioxane (5 mL). The reaction mixture washeated at 95° C. for 36 hr. The mixture was cooled to room temperature,diluted with ethyl acetate, and washed sequentially with water,saturated sodium bicarbonate, and brine then dried over magnesiumsulfate, filtered, and concentrated under vacuum to dryness. The crudeproduct was purified by flush chromatograph on silica gel elute with 3:2hexane:ethyl acetate to afford compound 11 (174 mg) as a white solid.

Step 3: The compound 11 (148 mg, 0.33 mmol) was dissolved in 1 N sodiumhydroxide aqueous (3 ml) and methanol (10 ml) and the resulting solutionwas refluxed for 1 hr. Most of methanol was removed under vacuum. Waterwas added and mixture was sonicated and filtered. The collected solidwas dried over vacuum to afford 12 (82 mg) as white solid. Selected ¹HNMR data: (CD₃OD, 500 MHz) δ 5.48 (d, 1H, J=2.8 Hz), 4.04 (d, 1H, J=2.7Hz), 3.74 (dd, J=8.3 Hz, J=2.5 Hz), 3.56 (t, 1H, J=8.5 Hz), 3.43 (dt,1H, J=7.6 Hz, J=4.2 Hz), 1.25 (s, 3H), 0.75 (s, 3H). Melting Point:144-147° C.

(VI), 16α-bromoandrost-5-ene-3β-ol-11β-acetoxy-17-one (VII), (VIII),androst-5-ene-3β,11β,16α-triacetoxy-17-one (IX),androst-5-ene-3β,11β,16α-triacetoxy-17β-ol (X),androst-5-ene-3β,11β,16α,17β-tetrol (XI)

II. Compound I (4.0 g, 11.4 mmol) was dissolved in 100 ml anhydrous1,4-dioxane. Sodium methoxide (3.0 g, 55.2 mmol) was added and themixture was refluxed under anhydrous conditions for 3 hours withmonitoring by HPLC. Solvent was removed to ⅓ volume and mixture wasacidified w/ 2N HCl to pH=5-6. Mixture was extracted with 3×50 ml DCM.Organic layers were recombined and washed with 50 ml sat'd sodiumbicarbonate and 50 ml brine. After drying over sodium sulfate andevaporation of solvent, 3.56 g of 95% pure product were isolated.

III. Compound II (13.5 g) and p-toluenesulfonic acid (2.45 g) werestirred for 18 hours in 175 ml anhydrous acetic anhydride. The mixturewas then poured into 800 g of ice and stirred for 1 hour. Filtrationthrough a short silica gel plug gave 3.14 g of compound III.

IV. Compound III (100 mg) was dissolved in 1.55 ml ethylene glycol,followed by addition of triethyl orthoformate (3.77 ml) andp-toluenesulfonic acid (50 mg). The mixture was then refluxed for 1.5hours, and then poured into a hot mixture of 6 ml methanol and 0.08 mlpyridine. The mixture is cooled and 15 ml water was added. The mixturewas extracted with 3×30 ml ethyl acetate and washed with sat'd sodiumbicarbonate (20 ml) and brine (20 ml)). Drying over sodium sulfate andevaporation of solvent gave 50 mg of 97% pure V.

V. To a solution of 50 mg IV in 2 ml DMF was added a solution of 27 mgsodium borohydride dissolved in 0.5 ml water at room temperature. Thesolution was heated to 100° C. with vigorous stirring for 15 minutes,followed by cooling to room temperature. The reaction was poured into 18ml water, followed by 0.2 ml acetic acid. The mixture was extracted withethyl acetate (2×10 ml) and washed with sat'd sodium bicarbonate (10 ml)and brine (10 ml). Drying over sodium sulfate and evaporation of solventgave 39 mg of compound V.

VI. Compound V (50 mg) and p-toluenesulfonic acid (2 mg) were suspendedin a mixture of 2 ml acetone and 0.21 ml water refluxed for 1.5 hours.After evaporation of acetone, 10 ml water was added and productprecipitated out of solution. Filtration gave 42 mg of desired product95% pure.

VII. Compound VI (315 mg) and CuBr₂ (611 mg) were added to 7 mlanhydrous methanol and refluxed for 24 hours. The reaction mixture wasthen cooled and poured into 15 ml hot water and crude product isfiltered off. The crude product was then dissolved in 25 ml methanol/THF(1:1) and then 200 mg activated carbon was added. The solution wasboiled for 10 minutes and the carbon was filtered off. The crude productwas recrystallized from methanol to give 426 mg product of 75% purity.

VIII. Compound VII (420 mg) was dissolved in a mixture of 18 ml DMF and7 ml water. Aqueous sodium hydroxide was added while stirring (1N, 1.31ml). After 10 minutes, solution was poured into an ice/water mixturecontaining 1.5 ml 1M HCl. The solution was saturated with NaCl andextracted with 2×5 ml ethyl acetate. After drying over sodium sulfateand evaporation of solvent the crude product was purified by columnchromatography to give 300 mg of 95% pure VIII.

IX. Compound VIII (300 mg) was dissolved in 6 ml pyridine, followed byaddition of 0.34 ml acetyl chloride. The reaction was stirred for 18hours, and then poured into 30 ml ice water. The crude product wasfiltered off, then purified by column to give 180 mg pure product.

X. Compound IX (120 mg) was dissolved in 5 ml methanol and cooled in anice bath. Sodium borohydride (11.5 mg) was added over 5 minutes and theice bath was removed. After 1 hour the reaction was quenched with 0.2 mlacetic acid and 15 ml water was added. The mixture was extracted with3×20 ml ethyl acetate and washed with sat'd sodium bicarbonate (20 ml)and brine (20 ml). Drying over sodium sulfate followed by columnchromatography gave 86 mg of the desired product.

XI. Compound X (180 mg) was dissolved in 5 ml dry ethyl ether and cooledto −78° C. Methyl magnesium bromide was added dropwise (1.2 ml, 1M inethyl ether). The reaction was warmed to room temperature, the refluxedfor 3 hours. The reaction was then cooled and neutralized with 1M HCl.Precipitated product was filtered off and recrystallized 3 times frommethanol/water to give 36 mg of pure XI. Melting point=220.3-221.6° C.Selected NMR shifts: ¹H NMR (CD₃OD): 5.20 ppm (bs, 1H), 4.26 ppm (dd,J=3 Hz, 5 Hz, 1H), 3.88 ppm (m, 1H), 3.32 ppm (m, 1H), 3.19 ppm (d, J=8Hz, 1H), 1.24 ppm (s, 3H), 0.92 ppm (s, 3H).

Androst-5-ene-3β,16α-diacetoxy-7,17-dione (4),androst-5-ene-3β,16α-diacetoxy-7β,17β-diol (HE3467)

Synthesis of 2. To a solution of 1 (3.44 g, 10 mmol) and TMS-Cl (2.15ml, 16.5 mmol) in THF (100 ml) cooled to −78° C. was added 2.0 M LDA(7.5 ml, 15 mmol) dropwise. The solution was stirred for 30 min andwarmed to room temperature. The reaction mixture was partitioned between100 ml 1:1 hexane/ether and 100 ml water. The organic layer was washedwith brine (3×30 mL) and dried over Na₂SO₄. A yellow oil was obtainedafter solvent was removed. The crude was chromatographed silica gel with5-20% EtOAc/hexane to recover 1.9 g of 1 and 2 as a white solid (600 mg,1.54 mmol), 31% yield.

Synthesis of 3. To a solution of 2 (100 mg, 0.26 mmol) in THF (3 ml)cooled to 0° C. was added mCPBA (77%, 62.2 mg, 0.27 mmol) and warmed upto room temperature. 0.5N HCl (3 ml) was added and stirred for 20 min,extracted with ether. The extracts were were washed with saturatedsodium bicarbonate, brine, dried over Na₂SO₄. The product 3 was obtainedafter removing solvent (90 mg, 0.26 mmol), 100% yield.

Synthesis of 4. To a solution of 3 (721 mg, 2.0 mmol) in pyridine (10mL) cooled to 0° C. was added acetyl chloride dropwise and stirred at 0°C. for 2 hours. The reaction was quenched with water (300 mL) andstirred for 15 min. A solid was formed and collected by filtration. Thesolid was washed with water, 1N HCl and water, and dried under vacuum toafford an off white solid 4 (737 mg), 90% yield.

Synthesis of HE3467. To a solution of 4 (300 mg, 0.75 mmol) in 1:1MeOH/THF (15 ml) cooled to −15° C. was added NaBH₄ (42.5 mg, 1.12 mmol)over 15 min. A solution of cerium chloride (300 mg, 0.81 mmol) in MeOHcooled −15° C. was added and stirred for 2 min. The reaction wasquenched with 1N HCl then poured to 90 mL water. A solid was formed andcollected by filtration. The solid was washed with 1N HCl and water,dried under vacuum to afford HE3467 (183 mg, 0.45 mmol), 60% yield. ¹HNMR (CD3OD): δ 5.29 (s, 1H), 4.89 (m, 1H), 4.55 (m, 1H), 3.74 (d, 1H,J=6.52), 3.60 (d, 1H, J=4.76), 2.36 (d, 2H, J=1.27), 2.15-2.18 (m, 2H),2.05 (s, 3H), 2.01 (s, 3H), 1.9-1.1 (m, 11H), 1.11 (s, 3H), 0.82 (s,3H).

5α-Androstane-2β,3α,16α,17β-tetrol (22)

Step 1: To a solution of 13 (50.0 g, 0.172 mol) in pyridine (150 mL) wasadded p-toluensulfonyl chloride (47.0 g, 0.24 mol) at 0° C. The reactionmixture was stirred at 0° C. for 2 hr and then stirred at roomtemperature overnight. Water was added. The resulting precipitate wascollected by filtration and washed with water. The crude product waspurified by recrystallization from methanol to afford 14 (75.2 g) as awhite solid.

Step 2: A mixture of compound 14 (75 g, 0.169 mol) in 2,4,6 collidine(200 mL) was refluxed for 5 hr. After cooling, water (500 mL) was addedand resulting precipitate was collected by filtration and washed withwater. The solid was recrystallized in methanol to give a crude product(42.5 g). The crude product (20.0 g) was dissolved in chloroform (113mL) and acetic acid anhydride (37 mL). To this solution was added asolution of concentrated sulfuric acid (3 mL) in chloroform (37 mL) and13 mL acetic acid anhydride at 0° C. The reaction mixture was stirred at0° C. for 0.5 hr then 700 mL water was added and stirred at roomtemperature for 6 hours. The resulting precipitate was collected byfiltration and washed with water, dried over vacuum to afford 15 (17.2g) as a white solid.

Step 3: The mixture of 15 (8.17 g, 0.030 mol) and copper bromide (10.8g, 0.046 mol) in anhydrous methanol (220 mL) was refluxed for 18 hr.After cooling, most of solvent was removed under vacuum and water (150mL) was added. The resulting precipitate was collected by filtration andwashed with water. The solid was recrystallized in methanol to afford 16as white solid (6.76 g).

Step 4: To the stirring solution of 16 (6.69 g, 0.019 mol) inN,N-dimethylformamide (180 mL) was added 1 N sodium hydroxide aqueoussolution (22 mL, 0.022 mol). The reaction mixture was stirred at roomtemperature for 0.5 hr. 1N aqueous hydrochloric acid (3 ml) and 100 mlwater were added. The resulting solution was extracted with ethylacetate (3×250 mL). The combined extracts were dried over magnesiumsulfate, filtered, and concentrated under vacuum to afford 17 (4.37 g)as a waxy solid.

Step 5: To a solution of 17 (3.74 g, 0.013 mol) in pyridine (20 mL) wasadded acetyl chloride (2.18, 0.028 mol) at 0° C. The reaction mixturewas stirred at 0° C. for 1 hr. The resulting mixture was warmed to roomtemperature and stirred for another 1 hour. Water was added. Theprecipitate was collected by filtration and washed with water. The solidwas dried over vacuum to give 18 (4.25 g) as a white solid.

Step 6: To a solution of 18 (2.4 g, 0.0072 mol) in methanol (80 mL) wasadded Sodium borohydride (1.2 g, 0.031 mol) at 0° C. The reactionmixture was stirred at 0° C. for 1 hr. The reaction was quenched by theaddition of acetic acid (6 mL) and water (15 mL). Most of methanol wasremoved under reduced pressure. The residual sludge was partitionedbetween ethyl acetate (80 mL) and water (20 mL). The organic layer waswashed with 1N aqueous hydrochloric acid, neutralized with saturatedaqueous sodium bicarbonate solution and then dried over magnesiumsulfate, filtered, and evaporated to afford crude product 19 (1.92 g) asa white solid.

Step 7: To a solution of 19 (1.9 g, 0.0057 mol) in pyridine (20 mL) wasadded acetyl chloride (1 mL, 0.014 mol) at 0° C. The reaction mixturewas stirred at 0° C. for 1 hr. The resulting mixture was warmed to roomtemperature and stirred for another 1 hr then most of the solvent wasremoved under vacuum. The residual sludge was partitioned between ethylacetate (80 mL) and water (20 mL). The organic layer was washed with 1Naqueous hydrochloric acid, saturated sodium bicarbonate aqueous solutionthen dried over magnesium sulfate, filtered and evaporated to give acrude product. The crude product was purified by flash chromatography onsilica gel and eluted with 1:10 ethyl acetate:hexane to afford the 20(1.4 g) as a white solid.

Step 8: To a solution of 20 (980 mg, 2.61 mmol) in chloroform (25 mL)was added m-chloroperoxybenzoic acid (3.6 mmol). The reaction mixturewas stirred at room temperature for 2 hr. The organic layer was washedwith saturated sodium bicarbonate aqueous solution, washed with waterand then dried over magnesium sulfate, filtered and evaporated to give acrude product. The crude product was purified by flash chromatograph onsilica gel eluted with 1:10 ethyl acetate:hexane to afford 21 (780 mg)as a white waxy solid.

Step 9: The solution of 21 (625 mg, 1.61 mmol) in acetic acid (8 mL) wasrefluxed for 5 hr. After cooling, the solvent was removed under vacuumto give an oil, which was further dried over vacuum overnight. Theresulting waxy solid was dissolved in 2 N sodium hydroxide aqueous (8mL) and methanol (15 mL) and the reaction was refluxed for 1 hr.Methanol was removed under vacuum and water was added. The resultingprecipitate was collected by filtration and washed with water and hotacetone. The collected solid was dried over vacuum to affordandrostane-2β,3α,16α,17β-tetrol or 22 (295 mg) as a white solid.Selected ¹H NMR data: (CD₃OD, 500 MHz) δ 3.98 (d, 1H, J=4.8 Hz), 3.79(br, 1H), 3.74 (br, 1H), 3.35 (d, 1H, 3.6 Hz), 0.99 (s, 3H), 0.77 (s,3H). mp: 260-263° C.

As will be apparent, the compounds described above can be used toprepare other formula 1 compounds, e.g., other esters or ethers of thesecompounds. Intermediates in the preparation of the title compounds canalso be used in the methods described herein.

Example 23

The capacity of formula 1 compounds to treat multiple sclerosis wasevaluated in experimental autoimmune encephalomyelitis (EAE) essentiallyas described in example 6 above. The protocol for conducting the EAEanimal model was described in (D. Auci et. al., Ann. NY. Acad. Sci. USA,1051:730-42, 2005). Female SJL mice (6-8 week old, average body weightof 25 g) obtained from Charles-River were kept under standard laboratoryconditions (non specific pathogen germ free) with ad libitum food andwater and were allowed to adapt one week to their environment beforecommencing the study. Animals were randomized into six groups of sevenanimals each and were (1) mice treated with vehicle, (2) mice treatedwith SU5416 (Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone),(3) mice treated with 17β-ethynylandrost-5-ene-3β,7β,17α-triol, (4) micetreated with androst-5-ene-3α, 7β,16α,17β-tetrol, (5) mice treated withandrost-5-ene-3β,7β,16α,17β-tetrol, (6) mice treated with3α-trifluoromethyl-androst-5-ene-3β,17β-diol, (7) mice treated with17α-trifluoromethyl-androst-5-ene-3β,17β-diol and (8) mice treated with5α-androstane-3β,17β-diol-16-oxime. EAA was induced with 200 μL of a 1:1emulsion of 75 μg proteolipid protein (PLP) and 6 mg/mL Mycobacteriumtuberculosis H37RA in complete Freund's adjuvant (CFA). The 200 μLinjection was divided among four sites that drained into the auxiliaryand inguinal lymphnodes. Pertussis toxin was used as a co-adjuvant andwas administered i.p. at 200 ng/mouse on day zero and day two postimmunization. Groups were treated with 0.1 mg of compound in 100 μLvehicle, or with vehicle alone, q.d. po (oral gavage) starting atclinical onset of disease and continuing through to day 30 postimmunization. Clinical onset is defined as the time when clinicalsymptoms of the disease attain a grading between 2-3 in 25% of the mice.Clinical grading was carried out by an observer unaware of thetreatment: 0=no illness, 1=flaccid tail, 2=moderate paraparesis,3=severe paraparesis, 4=moribund state, 5=death. Statistical analysisfor significant differences on clinical scores were performed by ANOVAfor unpaired data and to the non parametric Mann-Whitney test. A P value<0.05 was considered to be statistically significant. For statisticalanalysis, the mice that succumbed to EAE were assigned 5 only for theday of death and then were deleted from the experimental group.

As expected, classical signs of EAE developed in 8/8 (100%) of thevehicle-treated mice within day 19^(th) post immunization. The mean dayof onset was 15.5±3.9 (SD). In this group of animals the duration of thedisease was 12.3±4.3 days. The mean cumulative score from day 1 to 30was 24.8±7.8 and that from day 31 to day 54 (post treatment) was22.7±15.8. A course of EAE very similar to that observed in thevehicle-treated mice was observed in the animals treated with SU5416,androst-5-ene-3α,7β,16α,17β-tetrol and5α-androstane-3β,17β-diol-16-oxime, the so-treated mice exhibitingcumulative incidence of disease, duration of disease and mean cumulativeonset comparable to that of the controls. In contrast, the mice treatedwith androst-5-ene-3β,7β,16α,17β-tetrol,3α-trifluoromethylandrost-5-ene-3β,17β-diol or17α-trifluoromethylandrost-5-ene-3β,17β-diol exhibited a significantlyimproved course of EAE as compared to the vehicle-treated mice entailingsignificantly reduction of both one or more the mean cumulative scoreand duration. And in further contrast, neither of these 3 compoundssignificantly influenced the cumulative incidence of EAE or thelethality. Finally, although 17β-ethynylandrost-5-ene-3β,7β,17α-triolonly exhibited a trend toward reduced cumulative score and duration vsthe vehicle-treated mice, the effects appeared to be biologicalimportant (14.9±17.6 and 7±7.9 vs 24.8±7.8 and 12.3±4.3). The lack ofstatistical significance with this compound is probably due to the largenumber of mice being assigned score 0 throughout the observation periodwhich therefore resulted in a high standard deviation.

At the end of the treatment on day 30^(th), the mice were monitored forup to additional 24 days. It was possible to observe the diseasebecoming chronic in the vehicle-treated mice with cumulative scorescomparable to that of the treatment period. A substantial increase inthe cumulative score during the follow-up period as compared to thetreatment period was observed with SU5416 that passed from a meancumulative score of 25.5±8.9 to 35.5±13.2 and more modestly with17β-ethynylandrost-5-ene-3β,7β,17α-triol that passed from a meancumulative score of 14.9±17.6 to 18.4±20.6. In the mice treated with17β-ethynylandrost-5-ene-3β,7β,17α-triol t it was also possible toobserve an increase of the EAE incidence from 57.1% at the end of thetreatment period to 85.7% at the end of the follow-up period. On theother hand, the other compounds have appeared to maintain a similarcumulative score in the follow-up period as in the treatment period.This was particularly remarkable for3α-trifluoromethylandrost-5-ene-3β,17β-diol that passed from a meancumulative score of 11.2±4.8 during the treatment period to 10.8±10.3 atthe end of the follow-up period.

These results show that 17β-ethynylandrost-5-ene-3β,7β,17α-triol,androst-5-ene-3β,7β,16α,17β-tetrol,3α-trifluoromethylandrost-5-ene-3β,17β-diol and17α-trifluoromethyl-androst-5-ene-3β,17β-diol exerted powerfulanti-inflammatory properties in the PLP-induced model of EAE in SJLmice. Of particular relevance for the translation of these findings tothe clinical setting are the observations that the compounds are activein this EAE model even when given in a protocol starting on day 12^(th)post immunization when 24% of the mice had already developed clinicalsigns of EAE. Of particular note is the finding that SU5416 wasineffective in this setting. It has been previously reported that SU5416is effective in EAE (L. Bouerat et al., J. Med. Chem. 48: 5412-5414,2005). However, to obtain this result, the SU5416 compound wasadministered at the same time the animals were immunized. By contrast,in this protocol compounds such as17β-ethynylandrost-5-ene-3β,7β,17α-triol were not administered to theanimals until after disease symptoms were apparent, which shows that thecompounds can be used to effectively treat existing disease and toprevent or delay disease onset.

Example 25

Treatment of ionizing radiation exposure. The effect of selected F1Cs onsurvival of lethally-irradiated female B6D2F1 mice were compared tocontrol animals treated with vehicle alone. The animals were exposed to10 Gy of total body irradiation at 2.5 Gy/min using a ¹³⁷Cs source.Groups of 12 animals were used in a total of 5 groups. For Groups 1, 2,3, and 5, test article was administered as a 100 μL volume, bysubcutaneous injection, for three consecutive days, with the first doseadministered 2 to 4 hours following exposure to radiation. For Group 4,test article was administered as a 50 μL volume, by intramuscularinjection for three consecutive days. The formulation was a suspensioncontaining 0.1% w/v carboxymethyl-cellulose, 0.9% w/v sodium chlorideand 0.05% v/v phenol. The formulations were agitated to uniformlyresuspend the F1C before syringing, and injected into animals within afew minutes of drawing into the syringe to prevent settling in thesyringe.

The groups of animals were treated as follows. Group 1 received vehicleonly by daily subcutaneous injection for 3 consecutive days. Group 2received 0.6 mg in 100 μL of a suspension of3β,17β-dihydroxyandrost-5-ene by daily subcutaneous injection for 3consecutive days. Group 3 received 3.0 mg in 100 μL of a suspension of3β,17β-dihydroxyandrost-5-ene by daily subcutaneous injection for 3consecutive days. Group 4 received 0.6 mg in 50 μL of a suspension of3β,17β-dihydroxyandrost-5-ene by daily intramuscular injection for 3consecutive days. Group 5 received 0.6 mg in 100 μL of a suspension of3β-hydroxy-17β-aminoandrost-5-ene by daily subcutaneous injection for 3consecutive days. Survival of the animals was monitored for 21 daysafter irradiation and the following results were obtained. The number ofsurviving animals is shown for day 6, 7, 12 and 21.

Day Group 6 7 12 21 1 vehicle control 12 11 4 1 2 0.6 mg s.c. 12 11 10 73 3.0 mg s.c. 12 12 9 7 4 0.6 mg i.m. 12 12 11 9 5 0.6 mg s.c. 12 12 1211

Example 25

Treatment of gastrointestinal inflammation. The capacity of formula 1compounds to limit or inhibit inflammation or symptoms of inflammationwas shown using an animal model for inflammatory bowel disease. Groupsof 3 male Wistar rats (180±20 grams) fasted for 24 hours before2,4-dinitrobenzene sulfonic acid (DNBS) or saline challenge were used.Distal colitis was induced by intra-colonic instillation of 0.5 mL of anethanolic solution of DNBS (30 mg in 0.5 mL of a 30% ethanol in salinesolution) after which 2 mL of air was injected through the cannula toensure that the solution remained in the colon. The volume used was 0.1mL per injection of 2 and 20 mg/mL of compound such asandrost-5-ene-3β,7β,17β-triol in a liquid formulation, which wasadministered by subcutaneous injection once a day for 6 days (0.2mg/animal/day or 2.0 mg/animal/day). The formulation contained 100 mg/mLof compound in a non-aqueous suspension, e.g., 2% benzyl alcohol w/v,0.1% Brij 96 w/v and equal volumes of PEG 300 and propylene glycol.Concentrations of 2 mg/mL and 20 mg/mL were obtained by diluting the 20mg/mL formulation with vehicle that lacked compound.

The first dose was given 30 minutes after DNBS challenge. Sulfasalazine(30 mg/mL in 2% Tween 80 in distilled water) was administered orally(PO) once a day (10 mL/kg/day) for 7 days, the first two doses beginning24 hours and 2 hours before DNBS challenge. The presence of diarrhea wasrecorded daily by examining the anal area. Animals were fasted for 24hours prior to being sacrificed. Animals were sacrificed on day 7 or day8 and their colons are removed and weighed. Before removal of the colon,signs of adhesion between the colon and other organs are recorded. Also,the presence of ulcerations was noted after weighing of each colon. The“net” change of colon-to-body weight (BW) ratio is normalized relativeto saline-challenged baseline group. A 25-30% decrease in “net”colon-to-body weight ratio was considered significant. The resultsshowed that androst-5-ene-3β,7β,17β-triol had a modest effect on thecourse of disease (about 15-20% decrease in net colon-to-body weightratio), while treatments with 17α-ethynylandrost-5-ene-3β,7β,17β-triolor androst-5-ene-3β,7β,16α,17β-tetrol were effective (about 25-35%decrease in net colon-to-body weight ratio).

Variations of this protocol include administration of compounds in anaqueous solution of 30% sulfobutylether-cyclodextrin in water using doselevels described above and/or one or more of 0.05 mg/animal/day, 0.1mg/animal/day, 0.5 mg/animal/day and 1.0 mg/animal/day.

Example 26

Treatment of neuron loss associated with trauma and osteoporosis or boneloss conditions. Immune competence is a complex function that can beacutely impaired following trauma-induced elevations in endogenousglucocorticoid (GC) levels. The compound 5-androstene-3β,7β,17β-trioladministered parenterally was used to preserve these immune function byexerting a trophic or anabolic activity. In an animal model of acutecerebral ischemic stroke consecutive to bilateral carotid occlusion ingerbils, treatment with 5-androstene-3β,7β,17β-triol significantlyimproved cognitive abilities when compared to stroke alone (p=0.03).Thus, the measured food-searching latency period in each group was6.9±0.9 seconds (sec) for sham, 46.9±13.6 sec for stroke alone and14.8±4.8 sec for stroke treated with 5-androstene-3β,7β,17β-triol.Concomitantly, the stroke-induced loss in CA1 hippocampal neuron countwas markedly abrogated by 5-androstene-3β,7β,17β-triol(sham=362,247±6,839; stroke=152,354±11,575; andstroke+5-androstene-3β,7β,17β-triol=207,854±47,334).

In bone loss conditions, 5-androstene-3β,7β,17β-triol affected theprincipal bone structures, i.e., cortical and trabecular layers and thegrowth plate. In thermally-injured mice (20% total body surface area)treated with 5-androstene-3β,7β,17β-triol, loss of cortical (femur) andtrabecular/cancellous (tibia) bone mass, as well as suppression ofchondrocyte proliferation in proximal tibial epiphyseal growth plate,were all significantly (p<0.01) prevented by5-androstene-3β,7β,17β-triol treatment. Histomorphometry of the femurcortical bone suggested an increase in bone formation rate. We observedpartial protection against loss of bone mineral content as measured bydual X-ray absorptiometry. The femur ash weight was significantly(p<0.01) greater than that in the vehicle-treated burned mice, showingthat 5-androstene-3β,7β,17β-triol preserved bone mineral content.Pro-inflammatory effects of chronically high GC levels in brain, suggestthat elevated GC levels worsen the outcome of neurological insults. Theadrenal steroid DHEA (5-androstene-3β-hydroxy-17-one), an upstreammetabolic precursor of 5-androstene-3β,7β,17β-triol, has beendemonstrated to prevent dexamethasone-induced thymic involution in mice(K. L. Blauer et al., Endocrinology, 129:3174, 1991). Taken together,these findings showed that 5-androstene-3β,7β,17β-triol suppressedGC-induced loss of functional nerve tissue and preserved bone structureafter thermal injury.

The capacity of compounds including 5-androstene-3β,7β,17β-triol,17α-ethynyl-5-androstene-3β,7β,17β-triol and 4-estrene-3α,17β-diol toreverse adverse effects of glucocorticoids in bone growth was shown inthe human MG-63 osteosarcoma cell line. MG-63 cells are osteoblasts,which are cells that mediate bone growth. This cell line has been usedextensively to study bone biology and to characterize the biologicalactivity of compounds for treatment of bone loss conditions (e.g., B. D.Boyan et al., J. Biol. Chem., 264(20):11879-11886, 1989; L. C. Hofbaueret al., Endocrinology, 140(10):4382-4389, 1999). Adverse toxicitiesassociated with elevated glucocorticoid levels include a decrease in theproduction of IL-6 and IL-8 by osteoblasts, including the MG-63 cellline, and an increase in the expression of the 11β-hydroxysteroiddehydrogenase type 1 enzyme (11β-HSD). Increased 11β-hydroxysteroiddehydrogenase type 1 enzyme results in increased levels of endogenousglucocorticoid activity by converting endogenous cortisone to the activecortisol, which inhibits bone growth. The 11β-HSD enzyme is expressed inliver, adipose tissue, brain and bone tissues. Cortisol generated by11β-HSD-1 contributes to osteoporosis, insulin resistance, type 2diabetes, dyslipidemia, obesity, central nervous system disorders suchas stroke, neuron death, depression and Parkinson Disease. Decreases inIL-6, IL-8 and osteoprotegerin are associated with decreased bone growthby osteoblasts. Pilot studies showed that the IC₅₀ for inhibition ofIL-6 from MG-63 cells by dexamethasone was 10 nM and the IC₅₀ forinhibition of growth of MG-63 cells by dexamethasone was 15.3 nM.

In this protocol, MG-63 cells were grown in the presence or absence ofthe synthetic glucocorticoid dexamethasone at a 30 nM concentration andin the presence or absence of formula 1 compound at 10 nM. Compound 1 inthe table below was 5-androstene-3β,7β,17β-triol, compound 2 was17α-ethynyl-5-androstene-3β,7β,17β-triol and compound 3 was4-estrene-3α,17β-diol. The results for these compounds are shown below.

MG-63 growth IL-6 IL-8 11β-HSD mRNA osteoprotegerin conditions pg/mLunits units pmol/L vehicle control 6.2 0.90 0.25 445 dexamethasone 1.30.12 1.0 280 compound 1 4.0 0.53 0.73 — compound 2 2.8 0.50 0.54 —compound 2 (1 nM) — — — 455 compound 3 4.1 0.55 0.75 —

These results showed that the compounds at 10 nM partially reversed theadverse effects of dexamethasone at 30 nM, which shows that thecompounds can reverse multiple toxicities associated with elevatedglucocorticoid levels in osteoblasts, which are the cells that mediatebone growth. In a related protocol, the compound17α-ethynyl-5-androstene-3β,7β,17β-triol at 1 nM also completelyreversed the decrease in osteoprotegerin synthesis by MG-63 cells aftergrowth of the cells for 7 hours in the presence of 30 nM dexamethasoneas shown in the table above. Osteoprotegerin is a factor associated withbone growth and decreased osteoprotegerin synthesis is associated withbone loss. Other compounds that completely or partially reversed thedecrease in osteoprotegerin synthesis by MG-63 cells in the presence of30 nM dexamethasone were17α-trifluoromethyl-5-androstene-3β,7β,17β-triol (normal or basalosteoprotegerin levels at 1 μM compound compared to vehicle control withno compound or dexamethasone), 5-androstene-3β,7β,16α,17β-triol (normalosteoprotegerin levels at 0.1 μM),3β,7α,16α,17β-tetrahydroxyandrost-5-ene (near normal osteoprotegerinlevels at 10 nM), 3α,7β,16α,17β-tetrahydroxyandrost-5-ene (normalosteoprotegerin levels at 10 nM),17α-methylandrost-5-ene-3β,17β-diol-7-one (increased osteoprotegerinlevels at 100 nM), 17α-methylandrost-5-ene-3β,7β,17β-diol (normalosteoprotegerin levels at 10 nM). Other compounds that partiallyreversed the decrease in osteoprotegerin in the presence of 30 nMdexamethasone included androst-5-ene-3β,17β-diol-7-oxime.

In similar protocols the compound 3α,17β-dihydroxyandrost-4-ene showedstatistically significant reversal of dexamethasone-induced suppressionof IL-8 and IL-6 by MG-63 cells and a decrease in dexamethasone induced11β-HSD mRNA.

To show that relevant effects could be obtained in vivo, the compound17α-ethynyl-5-androstene-3β,7β,17β-triol was administered to mice thatwere also treated daily with dexamethasone for 23 days to reduce levelsof osteoprotegerin in the animals. Osteoprotegerin levels in mice thatwere treated with vehicle and dexamethasone at 10 μg/day (positivecontrol group) had 3.3 pMol/L osteoprotegerin, while animals treatedwith vehicle, dexamethasone and 17α-ethynyl-5-androstene-3β,7β,17β-triolat 4 mg/kg/day had 6.4 pMol/L osteoprotegerin (p<0.05).

The degree of apoptosis of osteoblasts and osteocytes in murinevertebral bone as a function of estrogen deficiency was examined. SwissWebster mice (four months old) were ovariectomized. Twenty-eight dayslater, the animals were sacrificed, vertebrae were isolated, fixed andembedded, and then undecalcified in methacrylate. The prevalence ofosteoblast and osteocyte apoptosis was determined by the TUNEL methodwith CuSO₄ enhancement, and was found to be increased following loss ofestrogen. Treatment with a reference compound such as 17β-estradiol andwith F1Cs such as 4-estrene-3α,17β-diol and or17α-ethynyl-5-androstene-3β,7β,17β-triol were found to reduce apoptosis,which is consistent with reduced lone loss.

Collectively, the results described in this example are evidence thatcompounds such as 17α-ethynyl-5-androstene-3β,7β,17β-triol affect bonetissue by both increasing bone growth and by inhibiting bone loss.Compounds such as 17α-ethynyl-5-androstene-3β,7β,17β-triol and5-androstene-3β,7β,16α,17β-tetrol do not interact with androgenreceptor, estrogen receptor-α or estrogen receptor-β, which isconsistent with their capacity to treat bone loss conditions withoutexerting unwanted sex hormone activity.

Example 27

A thermal injury model using mouse ear tissue was used to characterizecompounds for their capacity to treat inflammation associated withthermal trauma. The conditions were the minimal burn injury whichprogressed to tissue necrosis in the exposed ear of untreated mice by24-72 hours post-burn. Groups of Balb/c mice, approximately nine weeksold were given an identifying mark and then divided into control andtreated subgroups. The thickness of the ear to be immersed in hot waterwas recorded, and then the entire ear of the anesthetized mouse wasdipped into 52° C. water for 24 seconds. Each mouse was returned to itscage after an injection of either the propylene glycol vehicle (control)or 100 mg of compound in propylene glycol. Ear swelling changes weremonitored on individual mice at pre-burn, and at various times afterthermal injury. Ear swelling changes were monitored on individual miceat pre-injury and at 1, 3, 6, 9, 12, 18, 24 and 48 hours after thermalinjury. Animals were treated with 100 mg of dehydroepiandrosterone(DHEA) dissolved in propylene glycol. Analysis of edema formation andresolution in control and DHEA-treated mice showed peak ear swelling, asa measure of edema, in both DHEA-treated and untreated burned mice atsix hours after injury.

In the untreated control group, the extent of swelling started todecline within 12 hours, and continued to decline rapidly over thesubsequent 12 hour periods. Between 24 and 48 hours post-burn, eartissue showed loss of from the micro-vascular occlusion of the originalzone of stasis. The compounds androst-5-ene-3β,17β-diol and16α-bromodehydroepiandrosterone protected treated animals against muchof the ischemic consequences of thermal injury to the ear. The compounds16α-hydroxydehydroepiandrosterone was less protective, i.e., it reducedthe extent of, but did not totally prevent progressive ischemia, and16α-chlorodehydroepiandrosterone was only slightly protective againstprogressive ischemia.

The effect of compounds on hemorrhagic shock and ischemia was examinedin another protocol. CF-1 mice at an age of 6-8 months were anesthetizedusing methoxyfluorothane and prepared for abdominal surgery. Each mousewas tested for the level of respiration, eye blink response and responseto a skin pinch to ensure a level of anesthesia appropriate for surgery.The duration of abdominal surgery was approximately two hours, duringwhich time 35-40% of the animal's blood volume is removed over a30-minute period. The removal of blood in a controlled manner simulatesthe effect of hemorrhagic shock. A slow intravenous infusion of theremoved blood and a 2× volume of resuscitation fluid (lactated Ringerssolution) into a central vein was made. The resuscitation fluid wassupplemented with either 2 mg dehydroepiandrosterone-3β-sulfate or theexcipient as a placebo. The peritoneum and overlying skin were suturedseparately. Animals were maintained at 38°-39° C. until recovery iscomplete. Under these conditions, most of the placebo-treated animalsdied within 24-48 hours. Four hours after surgery, a colony forming unit(CFU) assay for bacteria was performed and malondialdehyde in liver wasassayed using conventional techniques. Mesenteric lymph nodes (MLN) wereremoved and cultured on blood agar plates and the number of CFUs countedfollowing culturing. The liver was removed and the amountmalondialdehyde was measured. Treatment withdehydroepiandrosterone-3β-sulfate resulted in survival of 15/15 micewhile 1/15 vehicle control animals survived.

The effect of treatment in a rat model of hemorrhagic trauma wasexamined. Twenty-four rats were subjected to 40% loss of total bloodvolume, consisting of catheterization and laparotomy (soft tissueinjury) to mimic trauma and hemorrhage. One hour after onset ofhemorrhage, the animals were resuscitated with crystalloid fluid andpacked red blood cells (PRBCs). Twelve animals received one subcutaneousinjection of androst-5-ene-3β,7β,17β-triol in a methyl cellulosesuspension at a concentration of 40 mg/kg body weight in 100 μL/kg bodyweight, one hour after initiation of hemorrhage, but prior to fluidresuscitation. Twelve animals received subcutaneous methyl cellulosecontrol injection at 100 μL/kg body weight. Three days after inductionof hemorrhage, the twelve animals that receivedandrost-5-ene-3β,7β,17β-triol had a 100% survival rate; whereas themortality rate was 25%, in the untreated group (P<0.04, Barnard'sunconditional test of superiority using difference of two binomialproportions).

A reduced blood pressure hemorrhagic trauma protocol was also conductedas a second model of hemorrhagic trauma. In this protocol, 15 rats werehemorrhaged described above to a mean arterial pressure of about 35-40mmHg and resuscitated one hour from onset of the hemorrhage withcrystalloid and PRBCs. Seven animals received one animals received onesubcutaneous injection of androst-5-ene-3β,7β,17β-triol in a methylcellulose suspension at a concentration of 40 mg/kg body weight in 100μL/kg body weight, one hour after initiation of hemorrhage, but beforefluid resuscitation. Eight animals received subcutaneous methylcellulose control injection at 100 μL/kg body weight. Two days afterinduction of hemorrhage, mortality in the untreated group (n=8) was 75%.The mortality rate in the androst-5-ene-3β,7β,17β-triol-treated animalswas 43%, demonstrating that the compound was protective in cases ofhemorrhagic trauma where blood pressure was reduced.

Example 28

Metabolic stability. The metabolic stability of selected compounds wasexamined in vitro using microsomes obtained from liver tissue accordingto the following protocol. Microsomes in this protocol are capable ofhydroxylation reactions and redox reactions that interconvert hydroxyland ketones on the steroid molecules. Microsomes do not mediateconjugation reactions, e.g., sulfation of 3β-hydroxyl groups orglucuronidation of 3α-hydroxyl groups.

The protocol was performed as follows. (1) Prepared 0.5 mM compound inacetonitrile/water 35:65. For androst-5-ene-3β,17β-diol, prepared 0.145mg/mL, or 29.0 μL of a 1 mg/mL stock plus 171 μL solvent. For thestandard curve dilutions of the 0.5 mM stock was used to obtain finalconcentrations of androst-5-ene-3β,17β-diol at 10 μM, 5 μM and 1 μM. (2)Set up samples as follows. Each assay consisted of anandrost-5-ene-3β,17β-diol control and 1-8 unknown compounds. Tubes foreach compound was follows: 1-0′ 2-0′ 3-0′ 4-0′*5-0′*6-5 μM 7-1 μM 8-30′9-30′ 10-30′ where * designated denatured microsome negative controlreaction tubes. For additional compounds numbering was started at 11,21, 31, etc. (3) Added 315 μL PBS (pH 7.3-7.5) to each tube. Added 10 μLof the appropriate test article solution to each tube. (4) The internalstandard/acetonitrile solution. (5) The NADPH regenerating system (NRS)was 125 μL per tube. To PBS added 1.7 mg/ml NADP, 7.8 mg/mlglucose-6-phosphate, 6 units/mL glucose-6-phosphate dehydrogenase. FreshNRS for each experiment was kept on ice until use. (6) Each reactionused 125 μL of NRS in each tube. (7) Removed liver microsome preparationfrom −80° C. freezer and thawed in a room temperature water bath. Themicrosomal preparation was at a concentration of 20 mg/ml. Each reactionused 0.25 mg/tube and was diluted to a concentration of 5 mg/ml in PBS(i.e. 4-fold dilution) and kept on ice. (8) For the zero-time anddenatured microsome control tubes 500 μL acetonitrile at −20° C. wasadded. Zero time tubes were transferred to ice and denatured microsomecontrols were preincubated at 37° C. for 5 minutes. (9) Assay tubescontaining the microsomal preparation was also preincubated for 5 min at37° C. (10) For each incubation tube, the reaction was started byaddition of 50 μL of the microsome preparation and vortexing to mix.(11) Each reaction was terminated by adding 500 μL acetonitrile at −20°C. and vortexing. (12) After the reaction was terminated, 100 μL fromeach reaction tube was transferred to a fresh tube and 200 μL of waterand 1400 μL of methyl-t-butyl ether was added to each tube. The tubeswere Vortexed and centrifuged at 13,000 rpm for 10 min on a microfuge.The tubes were then put on a dry ice-methanol bath until aqueous layerwas frozen solid. (13) The methyl-t-butyl ether was transferred fromeach tube to a fresh tube and the solvent was evaporated ether undernitrogen and the precipitate was then resuspended in 100 μLacetonitrile/water 35:65 and analyzed by LCMS. Results are shown in thetable below for the incubation times shown below.

parent parent remaining remaining human mouse Compound microsomesmicrosomes androst-5-ene-3β,17β-diol 39% (10 min) 25% (10 min)androst-5-ene-3β,17β-diol 30% (90 min) — androst-5-ene-3β,7β,17β-triol86% (90 min) 89% (10 min) 17α-ethynylandrost-5- — 86%* (30 min)ene-3β,7β,17β-triol androst-5-ene-3β,7β,16α,17β- 100% (10 min) 100% (10min) tetrol androst-5-ene-3α,7β,16α,17β- 100% (10 min) 100% (10 min)tetrol androst-5-ene-3α,7α,16α,17β- 100% (10 min) 100% (10 min) tetrolandrost-5-ene-3β,7α,16α,17β- 100% (10 min) 100% (10 min) tetrol *ratmicrosome instead of mouse preparation

The results show that the tetrol compounds were resistant to redoxreactions, which is consistent with a greatly reduced degree ofmetabolism compared to the androst-5-ene-3β,17β-diol reference compound.This observation was quite unexpected because each of the four hydroxylgroups could potentially be reduced to a ketone, but none was in factaffected. Other compounds that were examined includedandrost-5-ene-3β,16α,17β-triol, androstane-3β,16α-diol-17-one andandrostane-3α,16α,17α-triol, all of which were metabolized by microsomesat a rate similar to the androst-5-ene-3β,17β-diol reference compound.

Example 29

Measurement of drug absorption with CaCo-2 cells. This protocol was usedto measure the influx of compounds across a CaCo-2 cell monolayer.CaCo-2 cells are human cells with a polarized, highly differentiatedcell line demonstrating an intestinal absorptive cell phenotype (J.Hunter et al., J. Biol. Chem., 268(20):14991-14997, 1993). This cellline is used to study the rate at which various compounds cross the cellmonolayer. Typically, confluent monolayers of Caco-2 cells are used tomodel the intestinal epithelium and to obtain permeability coefficientsfrom the steady-state flux of test compounds. This can provideinformation about a compound's potential to be orally bioavailable.

In this protocol, the cells were maintained in medium at 37° C., using100 μL per well of warm medium in a sterile 50 ml tube. The cells weregrown on sterile 24-well plates with 600 μL of differentiation mediumper well. The wells contained a transwell insert to allow twocompartments per well. 100 μL of differentiation medium was carefullyadded into each well, touching the pipette tip to the side of well.Cells were incubated at 37° C., 5% CO₂, saturating humidity for 48 hoursto form a monolayer. For each plate, tubes were numbered with tubes 1-24for basolateral buffer to serve as a basolateral zero time point(T_(o)). Tubes 26 to 49 were apical buffer containing test article toserve as apical T_(o). Tubes 51-74 were the T₂₀ time point (20 minute),76-99 were the T₄₀ time point, 101-124 were the T₈₀ time point, 126-149were the T₁₂₀ time point, and 151-174 were T₁₂₀ apical samples for massbalance determination. Tubes 175-179 were the 5-point standard curve forCompound 1, tubes 180-184 were the standard curve for Compound 2 and soon to tubes 230-234 for Compound 12. Tubes 1-49 were placed in 4 rows inrack 1, 51-99 in rack 2, 101-149 in rack 3, 151-174 in rack 4, and175-234 in racks 5 and 6.

Buffers were prepared by removing 150 mL of transport buffer from afresh 1000 mL bottle (at pH to 7.4 with 1 N HCl). This buffer is‘basolateral’. The pH of the remaining 850 mL was adjusted to 6.5 with 1N HCL for the ‘apical’ buffer. 150 mL of apical buffer was placed in aseparate vessel, and the remaining 700 mL was used the for rinsing.Buffers were stored at 4° C. but used at room temperature for theprotocol.

After differentiation medium reached room temperature, about 20 mL waspoured into a small beaker. The probe was equilibrated in this mediumfor 15 min. 24-well plates were removed from the incubator and allowedto reach room temperature. Each well was measured by the probe byinserting the probe into the well without touching the cell monolayer;press the TEST button when the probe is close to the medium surface andthe reading will go from 0000 to a number when the probe touches thesurface; a reading >1000Ω was acceptable. The apical buffer was thendecanted from the transwell insert and the entire plate was rinsed in a1000 mL beaker containing rinse buffer to remove all differentiatingbuffer. The transwells were then placed into the T20 plates. 10 μM oftest compound and controls (carabamazapine MW 236; hydrochlorothiazideMW 351) was added in apical buffer by adding 0.1 μmol (e.g. 29 μl of a 1mg/ml androst-5-ene-3β,17β-diol reference solution) to 10 mL of apicalbuffer. 0.6 mL of basolateral buffer was then added to all wells.

A solution of 50 μg/ml 3α,7β,16α,17β-tetrahydroxyandrost-5-ene as aninternal standard was made by adding 150 μL of the compound (1 mg/mL inethanol) to 10 mL acetonitrile/water (25:75). Standard curves were madein basolateral buffer for each compound. The 10 μM apical buffer wasdiluted six fold when passing into the basolateral compartment, so thestandard curve was prepared at a six fold lower concentration.

Concentration Apical TA (10 μM) Baso Buffer 2 μM 120 480 1 μM 60 540 0.5μM 30 570 0.2 μM 12 588 0.05 3 597

600 μL of basolateral buffer was placed in tubes 1-24 for the T_(o)controls. 100 μL of apical buffer plus test article plus 500 μl apicalbuffer (so that concentration will be in standard curve range) was addedto tubes 26-49 to serve as apical T_(o). Place 100 μl apical buffer pluscompound on the apical side. The time that the transwell was placed inthe plate was taken as time zero (T_(o)). At T=20, the transwells weremoved to the T40 plate and 600 μL of sample from the T20 plate was addedto the appropriate tube. At T=40, the transwell was moved to the T80plate and 600 μl of sample was taken from the T40 plate to theappropriate tube. At T=80, move the transwell to the T120 plate. Pipette600 μl of sample from the T80 plate to the appropriate tube and so onfor the remaining time points. 100 μL of the apical buffer was added tothe appropriate tube for mass balance. Samples will immediatelyextracted immediately were placed in a freezer.

300 μL of each sample was transferred from the assay tube into a labeled2 mL tube, except for tubes 151-174 (which contained only 100 μL); 50 μlof these samples were transferred and added to 250 μL of basolateralbuffer (resulting in a 6-fold dilution). 20 μL of the3α,7β,16α,17β-tetrahydroxyandrost-5-ene internal standard was added toeach tube and 1500 μL of methyl-t-butyl ether was added to each tube.The tubes were vortexed, centrifuged in a microcentrifuge for 10 min.and placed in methanol/dry ice bath until frozen. Fresh tubes werelabeled and the methyl-t-butyl ether was decanted from each frozen tubeinto the fresh tube. The methyl-t-butyl ether was then evaporated undernitrogen and reconstituted in 120 μL acetonitrile/water (35:65) andanalyzed by LCMS. In the table below compound 1 was3β,7β,16α,17β-tetrahydroxyandrost-5-ene, compound 2 was17α-ethynylandrost-5-3β,7β,17β-triol, compound 3 was3α,17β-dihydroxy-17α-ethynylandrostane, compound 4 was 3α, 7β,16α,17β-tetrahydroxyandrost-5-ene, compound 5 was2β,3α,16α,17β-tetrahydroxyandrostane, compound 6 was3β,16α-diacetoxy-7β,17β-dihydroxyandrost-5-ene, compound 7 was3β-acetoxy-17α-ethynylandrost-5-7β,17β-diol, compound 8 was3β-acetoxyandrost-5-7β,16α,17β-triol and compound 9 was17α-ethynylandrost-5-3α,7β,17β-triol.

Conc. Cumulative (μM) basolateral % apical apical conc. (μM) transportedTotal % Compound @T₀ in 80 min in 80 min transported 1 2.195 0.017 0.0080.8% 2 1.911 0.470 0.246 24.6% 3 2.727 0.411 0.151 15.1% 4 1.664 0.0190.012 1.2% 5 1.817 0.162 0.089 8.9% 6 1.776 0.185 0.104 17.8% 7 1.7100.195 0.114 31.4% 8 1.724 0.123 0.071 15.9% 9 1.773 0.531 0.299 29.9%

Studies with the CaCo-2 cell line indicated that tetrol compounds suchas androst-5-ene-3β,7β,16α,17β-tetrol were not highly permeable andwould thus not be expected to be orally bioavailable. Despite that, thecompound androst-5-ene-3β,7β,16α,17β-tetrol was active as describedabove when administered orally to mice in a diabetes treatment model.Other protocols showed that the degree of sulfation and the degree ofglucuronidation for the tetrol compounds such as3β,7β,16α,17β-tetrahydroxyandrost-5-ene and 3α,7β,16α,17β-tetrahydroxyandrost-5-ene was low for tetrol compoundscompared to diols. This activity may have arisen at least partly fromthe low metabolism of tetrol compounds in vivo.

1. A method to identify a compound with a potential to treat orameliorate a metabolic disorder in a mammal, comprising selecting acompound that (i) does not activate one, two or three of PPAR-α, PPAR-γand PPAR-δ in human or mammalian cells in vitro by more than about 30%when compared to suitable negative control human or mammalian cells invitro; (ii) inhibits or decreases the transcriptional activity or levelof NF-κB by about 20-80% in human or mammalian cells in vitro whencompared to suitable negative control human or mammalian cells in vitro;(iii) when compared to a suitable negative control or normal control,(a) decreases the degree of hyperglycemia, (b) slows the progression ofhyperglycemia, (c) delays the onset of hyperglycemia, (d) decreases therate of macular degeneration, (e) delays the onset of maculardegeneration, (f) decreases the occurrence or incidence of vascularulcers, (g) decreases the severity of vascular ulcers, (h) increasesinsulin sensitivity, (i) decreases glucose intolerance, (j) slows theprogression or rate of loss of pancreatic β-islet cell numbers or theircapacity to secrete insulin, (k) increases pancreatic β-islet cellnumbers or their capacity to secrete insulin, (l) slows the rate ofweight increase in db/db mice or mice with diet induced obesity, (m)decreases elevated levels of triglycerides, (n) decreases elevatedlevels total blood or serum cholesterol, (o) decreases normal orelevated levels of LDL, VLDL, apoB-100 or apoB-48 in blood or serum, (p)increases normal or low levels of HDL or apoA1 in blood or serum, (q)decreases or normalizes an elevated level of a phase reactive protein,optionally C reactive protein or fibrinogen in blood or serum, (r)decreases or normalizes hemoglobin A_(1C), (s) decreases or normalizesfasting blood glucose levels, (t) normalizes serum or blood glucose inan oral glucose tolerance test, (u) normalizes serum or blood glucose ina 2 hour post prandial glucose test or (v) increases whole body ortissue glucose disposal or uptake in a human or another mammal in vivo,for 1, 2 or 3 of the foregoing; (iv) optionally, does not activate oneor more of a glucocorticoid receptor, an androgen receptor an estrogenreceptor-α, estrogen receptor-β or a biologically active variant of anyof these biomolecules in human or mammalian cells in vitro by more thanabout 30% when compared to suitable negative control human or mammaliancells in vitro; and (v) optionally inhibits the level or activity ofphosphoenolpyruvate carboxykinase (PEPCK) or a 11β-hydroxysteroiddehydrogenase (11β-HSD), optionally 11β-HSD type 1 or 11β-HSD type 2 orthe level of a mRNA that encodes PEPCK or a 11β-HSD, in hepatocytes orliver-derived cells in vitro or in liver cells or tissue obtained fromliver cells or tissue in vivo; wherein the compound with a potential totreat or ameliorate the metabolic disorder in a mammal is identified andoptionally recorded as such in a written or a readable electronicmedium.
 2. The method of claim 1 wherein the mammal is a rodent, anobese rodent, a human or an obese human.
 3. The method of claim 2wherein the metabolic disorder is type II diabetes, hyperglycemia,elevated nonesterified fatty acids or insulin resistance.
 4. The methodof claim 3 wherein the compound is selected from formula 1 compoundshaving the structure

wherein, the dotted lines are optional double bonds or if no double bondis present at the 4-5 or 5-6 positions, then hydrogen is present in theα- or β-configuration; one R¹ is —H or a carbon-linked moiety such asoptionally substituted alkyl and the other R¹ is an oxygen-linkedmoiety, a sulfur-linked moiety or a nitrogen-linked moiety, or both R¹together are ═O, ═NOH or ═NO—C₁₋₆ alkyl; one R² is —H or a carbon-linkedmoiety such as optionally substituted alkyl and the other R² is —H, anoxygen-linked moiety, a sulfur-linked moiety or a nitrogen-linkedmoiety, or both R² together are ═O; one R³ is —H or a carbon-linkedmoiety such as optionally substituted alkyl and the other R³ is —H, anoxygen-linked moiety, a sulfur-linked moiety or a nitrogen-linkedmoiety; one R⁴ is —H or a carbon-linked moiety such as optionallysubstituted alkyl and the other R⁴ is an oxygen-linked moiety, asulfur-linked moiety or a nitrogen-linked moiety, or both R⁴ togetherare ═O, ═NOH or ═NO—C₁₋₆ alkyl; one R⁵ is —H or a carbon-linked moietysuch as optionally substituted alkyl and the other R⁵ is —H, anoxygen-linked moiety, a sulfur-linked moiety or a nitrogen-linkedmoiety, provided that if a double bond is present at the 4-5 position,then R⁵ is —H, an oxygen-linked moiety, a sulfur-linked moiety or anitrogen-linked moiety; R⁶ is —H or C₁₋₆ optionally substituted alkyl,wherein R⁶ optionally is —CH₃; and R⁷ is —H or C₁₋₆ optionallysubstituted alkyl, wherein R⁷ optionally is —CH₃, —CH₂OH or —C₂H₅; R⁸ is—CH₂—, or —C(R¹⁰)₂— where R¹⁰ independently or together are —H, ═O, acarbon-linked moiety such as optionally substituted alkyl, anoxygen-linked moiety, optionally —OH or an ester or ether optionallyselected from —OC(O)—CH₃, —OC(O)—C₂H₅, —OCH₃ and —OC₂H₅ in the α- orβ-configuration or R⁸ is a sulfur-linked moiety or a nitrogen-linkedmoiety optionally selected from —SH, —SC(O)—CH₃, —SC(O)—O₂H₅, —SCH₃ and—SC₂H₅ in the α- or β-configuration; and R⁹ is —CH₂—, or —C(R¹⁰)₂— whereR¹⁰ independently or together are —H, halogen, ═O, a carbon-linkedmoiety such as optionally substituted alkyl, an oxygen-linked moiety,optionally —OH or an ester or ether optionally selected from —OC(O)—CH₃,—OC(O)—C₂H₅, —OCH₃ and —OC₂H₅ in the α- or β-configuration, or R⁸ is asulfur-linked moiety or a nitrogen-linked moiety optionally selectedfrom —SH, —SC(O)—CH₃, —SC(O)—C₂H₅, —SCH₃ and —SC₂H₅ in the α- orβ-configuration.
 5. The method of claim 4 wherein, the oxygen-linkedmoiety is —OH, an ester, phosphate, a phosphoester, sulfate, a sulfateester, amino acid, a peptide, an ether, a carbonate, a carbamate, or apolymer, any of which are in the α-configuration or the β-configuration;the sulfur-linked moiety is —SH, a thioester or a thioether, any ofwhich are in the α-configuration or the β-configuration; thenitrogen-linked moiety is —NH₂, an amino acid, a peptide, a carbamate,an amide, monosubstituted amine or a disubstituted amine, any of whichare in the α-configuration or the β-configuration, or thenitrogen-linked moiety is ═NOH or ═NO—C₁₋₆ alkyl, where the aminesubstitution(s) optionally are optionally substituted alkyl and providedthat there is 0 or one ═NOH or ═NO—C₁₋₆ alkyl moieties present; and thecarbon-linked moiety is optionally substituted alkyl, acyl or thioacyloptionally selected from the group consisting of ═CH₂, ═CHOH, —CH₃,—CF₃, —C₂H₅, —C₂F₅, —CH═CH₂, —CCH, —CCOH, —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OHand —C(O)CH₂-halogen.
 6. The method of claim 5 wherein the compound hasthe formula


7. The method of claim 5 wherein the compound has the formula


8. The method of claim 5 wherein the compound has the formula


9. The method of claim 5 wherein the compound has the formula


10. The method of claim 4 wherein R⁶ is —H or —CH₃.
 11. The method ofclaim 10 wherein one R² and R³ is —H, or C1-4 optionally substitutedalkyl and the other R² and R³ is —OH, an ester or an ether, wherein theester is optionally selected from the group consisting of —O—C(O)—CH₃,—O—C(O)—CF₃, —O—C(O)—CH₂CH₃ and —O—C(O)—(CH₂)₂CH₃, and/or wherein one R⁵is —H or C1-4 optionally substituted alkyl and the other R⁵ is —OH, —SHor an ester.
 12. The method of claim 11 wherein R⁴ in theβ-configuration is —OH, an ester or an ether and R⁴ in theα-configuration is or optionally substituted C₁₋₈ alkyl optionallyselected from the group consisting of —CH₃, —CF₃, —CN, —C₂H₅, —C₂F₅,—CH═CH₂, —CCH or both R⁴ together are ═NOH.
 13. The method of claim 4wherein the formula 1 compound is17α-ethynylandrost-5-ene-3β,7β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3α,7β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3β,7β,17β-triol,17α-ethynylandrost-5-ene-3β,7β,17β-triol,17β-ethynylandrost-5-ene-3β,7β,17α-triol,17α-ethynylandrost-4-ene-3β,7β,17β-triol,17α-ethynylandrostane-3β,7β,17β-triol,17α-ethynylandrost-5-ene-3α,7β,17β-triol,17α-ethynylandrost-4-ene-3α,7β,17β-triol,17α-ethynylandrostane-3α,7β,17β-triol,17α-ethynylandrost-5-ene-3β,7α,17β-triol,17α-ethynylandrost-4-ene-3β,7α,17β-triol,17α-ethynylandrostane-3β,7α,17β-triol,17α-ethynylandrost-5-ene-3α,7α,17β-triol,17α-ethynylandrost-4-ene-3α,7α,17β-triol,17α-ethynylandrostane-3α,7α,17β-triol,17α-ethynylandrost-5-ene-7β,17β-diol-3-one,17α-ethynylandrost-5-ene-3β,17β-diol-7-one,17α-ethynylandrost-5-ene-3α,17β-diol-7-one,17α-chloroethynylandrost-5-ene-3β,7β,17β-triol,17α-chloroethynylandrost-5-ene-3α,7β,17β-triol,17α-ethynylandrost-5-ene-3β,4β,16α,17β-tetrol,4-acetoxy-17α-ethynylandrost-4-ene-3β,16α,17β-triol,17α-ethynylandrost-5-ene-3α,4β,16α,17β-tetrol,4-acetoxy-17α-ethynylandrost-4-ene-3α,16α,17β-triol,17α-ethynylandrost-5-ene-3β,11β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3α,11β,16α,17β-tetrol,17α-ethynylandrost-5-ene-3β,11β,16β,17β-tetrol,17β-ethynylandrost-5-ene-3β,11β,16β,17α-tetrol,17α-ethynylandrost-5-ene-2β,3β,16α,17β-tetrol,17α-ethynylandrost-5-ene-2β,3α,16α,17β-tetrol,17α-ethynylandrost-5-ene-2α,3β,16α,17β-tetrol,17α-ethynylandrost-5-ene-2α,3α,16α,17β-tetrol, or an analog of any ofthese compounds wherein the hydroxyl group at the 3-position, ifpresent, is replaced with an ester, optionally selected from —OC(O)CH₃and —OC(O)C₂H₅.
 14. The method of claim 4 wherein the formula 1 compoundis 17α-ethynylandrost-5-ene-3β,7β,17β-triol.